Acyl-ACP Thioesterase

ABSTRACT

An acyl-ACP thioesterase consisting of an amino acid sequence of the 115 th  to 274 th  amino acids set forth in SEQ ID NO: 1; an acyl-ACP thioesterase gene encoding the protein; a transformant having the gene; and a method of producing a lipid using the transformant.

TECHNICAL FIELD

The present invention relates to a novel acyl-ACP thioesterase, and agene encoding the same. Further, the present invention relates to atransformant having the acyl-ACP thioesterase gene and a method ofproducing a lipid using the same.

BACKGROUND ART

Fatty acids are one of the principal components of lipids. In vivo,fatty acids are attached to glycerin via an ester bond to form lipidssuch as triacylglycerol. Many animals and plants store and utilize fattyacids as an energy source. These fatty acids and lipids are widelyutilized for food or industrial use.

For example, higher alcohol derivatives that are obtained by reducinghigher fatty acids having approximately 12 to 18 carbon atoms are usedas surfactants. Alkyl sulfuric acid ester salts and alkylbenzenesulfonicacid salts are utilized as anionic surfactants, and polyoxyalkylenealkyl ethers and alkyl polyglycosides are utilized as nonionicsurfactants. These surfactants are used for detergents or disinfectants.As other higher alcohol derivatives, cationic surfactants such asalkylamine salts and mono- or dialkyl-quaternary amine salts arecommonly used for fiber treatment agents, hair conditioning agents ordisinfectants, and benzalkonium type quaternary ammonium salts arecommonly used for disinfectants or antiseptics. Moreover, vegetable fatsand oils are used also as raw materials of biodiesel fuels.

Fatty acids and lipids are widely used for various applications shownabove. Therefore, it has been attempted to enhance the productivity offatty acids or lipids in vivo by using plants and the like. Further, theapplications and usefulness of fatty acids depend on the number ofcarbon atoms. Therefore, controlling of the number of carbon atoms ofthe fatty acids, namely, a chain length thereof has also been attempted.For example, a method of accumulating fatty acids having 12 carbon atomsby introducing an acyl-ACP thioesterase derived from Umbellulariacalifornica (California bay) (Patent Literature 1, and Non-PatentLiterature 1) has been proposed.

Attention has been recently focused on algae to the effect that thealgae are useful in biofuel production. The algae can produce lipidsthat can be used as the biodiesel fuels through photosynthesis, and donot compete with foods.

Therefore, the algae attract attention as next-generation biomassresources. Moreover, the algae are also reported to the effect that thealgae have higher lipid production and accumulation ability incomparison with plants.

Research has started on a lipid synthesis mechanism of the algae andproduction technologies applying the mechanism, but unclear parts remainin many respects. For example, almost no report has been made so far onthe above-mentioned acyl-ACP thioesterase derived from algae, either,and only limited examples of reports are made on Class Diatomea or thelike (for example, Non-Patent Literature 2).

CITATION LIST Patent Literature

-   Patent Literature 1: JP-A-7-501924 (“JP-A” means unexamined    published Japanese patent application)

Non-Patent Literatures

-   Non-Patent Literature 1: Voelker T A, Worrell A C, Anderson L,    Bleibaum J, Fan C, Hawkins D J, Radke S E, Davies H M., “Fatty acid    biosynthesis redirected to medium chains in transgenic oilseed    plants”, Science. 1992 Jul. 3; 257 (5066), p. 72-74.-   Non-Patent Literature 2: Yangmin Gong, Xiaojing Guo, Xia Wan, Zhuo    Liang,

Mulan Jiang, “Characterization of a novel thioesterase (PtTE) fromPhaeodactylum tricornutum”, Journal of Basic Microbiology, 2011December, Volume 51, p. 666-672.

SUMMARY OF INVENTION

The present invention is contemplated for providing a novel acyl-ACPthioesterase derived from algae, and an acyl-ACP thioesterase geneencoding the same. Further, the present invention is contemplated forproviding a transformant having the gene. Furthermore, the presentinvention is contemplated for providing a method of producing a lipidusing the transformant.

The present inventors made extensive studies so as to search a novelacyl-ACP thioesterase derived from algae. As a result, they found that anovel acyl-ACP thioesterase derived from algae belonging to the genusNannochloropsis and an acyl-ACP thioesterase gene encoding thethioesterase. The present invention was completed based on thesefindings.

The present invention relates to a protein selected from the following(a) to (c):

-   (a) A protein consisting of an amino acid sequence of the 115^(th)    to 274^(th) amino acids set forth in SEQ ID NO: 1;-   (b) A protein consisting of an amino acid sequence having 50% or    more identity with the amino acid sequence of the protein (a), and    having acyl-ACP thioesterase activity; and-   (c) A protein containing the amino acid sequence of the protein (a)    or (b), and having acyl-ACP thioesterase activity.    (Hereinafter, referred to as “the protein of the present invention”    or “the acyl-ACP thioesterase of the present invention”)

The present invention also relates to a gene encoding the protein of thepresent invention, preferably a gene consisting of any one of thefollowing DNAs (d) to (f):

(d) A DNA consisting of a nucleotide sequence of the 343^(rd) to825^(th) nucleotides set forth in SEQ ID NO: 2;(e) A DNA consisting of a nucleotide sequence having 50% or moreidentity with the nucleotide sequence of the DNA (d), and encoding aprotein having acyl-ACP thioesterase activity; and(f) A DNA containing the nucleotide sequence of the DNA (d) or the DNA(e), and encoding a protein having acyl-ACP thioesterase activity.(Hereinafter, referred to as “the gene of the present invention” or “theacyl-ACP thioesterase gene of the present invention”)

The present invention also relates to a transformant obtained byintroducing the gene of the present invention into a host.

(Hereinafter, referred to as “the transformant of the presentinvention”)

The present invention also relates to a method of producing a lipid,containing steps of:

culturing the transformant of the present invention in a medium; and

collecting a lipid from the resulting cultured product.

(Hereinafter, referred to as “the method of producing a lipid of thepresent invention”)

The present invention also relates to a method of modifying a fatty acidcomposition in a lipid, containing introducing the gene of the presentinvention into a host.

The present invention also relates to a method of enhancing productivityof a lipid, containing introducing the gene of the present inventioninto a host.

The present invention provides a novel acyl-ACP thioesterase and anacyl-ACP thioesterase gene encoding the same. The present invention alsoprovides a transformant having the acyl-ACP thioesterase gene. Further,the present invention provides a method of producing a lipid using thetransformant. The transformant and the production method of the presentinvention have the excellent productivity of lipids, and therefore theycan be suitably used for the industrial production of fatty acids orlipids.

Other and further features and advantages of the invention will appearmore fully from the following description.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be explained.

In the present invention, the term “lipid(s)” covers simple lipids suchas neutral lipids, wax, and ceramides; complex lipids such asphospholipids, glycolipids, and sulfolipids; and derived lipids such asfatty acids, alcohols, and hydrocarbons.

1. Acyl-ACP Thioesterase

The protein of the present invention includes a protein at least havingan amino acid sequence of the 115^(th) to 274^(th) amino acids set forthin SEQ ID NO: 1, and a protein functionally equivalent to the protein.Specifically, the protein of the present invention includes thefollowing proteins (a) to (c).

-   (a) A protein consisting of an amino acid sequence of the 115^(th)    to 274^(th) amino acids set forth in SEQ ID NO: 1.-   (b) A protein consisting of an amino acid sequence having 50% or    more identity with the amino acid sequence of the protein (a), and    having acyl-ACP thioesterase activity.-   (c) A protein containing the amino acid sequence of the protein (a)    or the protein (b), and having acyl-ACP thioesterase activity.

The protein consisting of the amino acid sequence set forth in SEQ IDNO: 1 is a novel acyl-ACP thioesterase derived from an alga belonging tothe genus Nannochloropsis, Nannochloropsis gaditana.

The acyl-ACP (acyl carrier protein) thioesterase is an enzyme involvedin the biosynthesis pathway of fatty acids and derivatives thereof (suchas triacylglycerol (triglyceride)). This enzyme hydrolyzes a thioesterbond of an acyl-ACP to form free fatty acids in a plastid such as achloroplast of plants and algae or in a cytoplasm of bacteria, fungi andanimals. The acyl-ACP is a composite composed of an acyl group as afatty acid residue and an acyl carrier protein, and is an intermediatein the process of fatty acid biosynthesis. The function of thethioesterase completes the synthesis of the fatty acid synthesis on theACP, and then the thus-produced free fatty acids are supplied to thesynthesis of triglyceride and the like. To date, several acyl-ACPthioesterases having different reaction specificities depending on thenumber of carbon atoms and the number of unsaturated bonds of acylgroup(fatty acid residue) of acyl-ACP substrate are identified. Therefore,they are considered to be an important factor in determining fatty acidcomposition of an organism.

In the present invention, the “having thioesterase activity” meanshaving an activity of hydrolyzing a thioester bond of an acyl-ACP.

One example of nucleotide sequences encoding the amino acid sequence setforth in SEQ ID NO: 1 is a nucleotide sequence set forth in SEQ ID NO:2. The gene consisting of the nucleotide sequence set forth in SEQ IDNO: 2 is derived from Nannochloropsis paditana. Genome sequence data ofNannochloropsis gaditana has been published in 2012 (see RandorRadakovits, et al., “Draft genome sequence and genetic transformation ofthe oleaginous alga Nannochloropsis gaditana”, Nature Communications,DO1:10.1038/ncomms1688, 2012).

The present inventors have identified the gene consisting of thenucleotide sequence set forth in SEQ ID NO: 2 as an acyl-ACPthioesterase gene. Further, they have identified an important region foracyl-ACP thioesterase activity in the amino acid sequence encoded by thegene.

A recombinant protein at least having an amino acid sequence of the115^(th) to 274^(th) amino acids set forth in SEQ ID NO: 1 acts as anacyl-ACP thioesterase as demonstrated in the working examples below.That is, it is thought that the region from 115^(th) to 274^(th) aminoacids is sufficient for acyl-ACP thioesterase activity, with respect tothe amino acid sequence set forth in SEQ ID NO: 1.

In the protein (b), the sequence identity of amino acid sequence ispreferably 60% or more, more preferably 70% or more, further preferably80% or more, further more preferably 90% or more, and particularlypreferably 95% or more, in view of acyl-ACP thioesterase activity.

In the present specification, the sequence identity of the amino acidsequence and nucleotide sequence is calculated through theLipman-Pearson method (see Science, 227, pp. 1435, (1985)).Specifically, the identity can be determined through use of a homologyanalysis (homology search) program of genetic information processingsoftware Genetyx-Win (Software Development Co., Ltd.) with the unit sizeto compare (ktup) being set to 2.

Specific examples of the protein (b) include the following proteins (a1)and (a2).

(a1) A protein consisting of an amino acid sequence of the 128^(th) to287^(th) amino acids set forth in SEQ ID NO: 14.(a2) A protein consisting of an amino acid sequence of the 126^(th) to285^(th) amino acids set forth in SEQ ID NO: 16.

The protein consisting of the amino acid sequence set forth in SEQ IDNO: 14 is a novel acyl-ACP thioesterase derived from Nannochloropsisoculata. The amino acid sequence of the 128^(th) to 287^(th) amino acidsset forth in SEQ ID NO: 14 has about 91% identity with the amino acidsequence of the 115^(th) to 274^(th) amino acids set forth in SEQ ID NO:1.

The protein consisting of the amino acid sequence set forth in SEQ IDNO: 16 is a novel acyl-ACP thioesterase derived from Nannochloropsispranulata. The amino acid sequence of the 126^(th) to 285^(th) aminoacids set forth in SEQ ID NO: 16 has about 90% identity with the aminoacid sequence of the 115^(th) to 274^(th) amino acids set forth in SEQID NO: 1.

Recombinant proteins at least having the amino acid sequence of the128^(th) to 287^(th) amino acids set forth in SEQ ID NO: 14 or the aminoacid sequence of the 126^(th) to 285^(th) amino acids set forth in SEQID NO: 16 act as an acyl-ACP thioesterase as demonstrated by the workingexamples below.

The protein (b) is preferably the protein (a1) or (a2).

Further, the protein (b) is also preferably a protein consisting of anamino acid sequence of the protein (a) in which one or several aminoacids (preferably 1 or more and 10 or less amino acids, more preferably1 or more and 5 or less amino acids, and further preferably 1 or moreand 3 or less amino acids) are mutated.

Further, the protein (b) is also preferably a protein consisting of anamino acid sequence of the protein (a1) in which one or several aminoacids (preferably 1 or more and 10 or less amino acids, more preferably1 or more and 5 or less amino acids, and further preferably 1 or moreand 3 or less amino acids) are mutated.

Further, the protein (b) is also preferably a protein consisting of anamino acid sequence of the protein (a2) in which one or several aminoacids (preferably 1 or more and 10 or less amino acids, more preferably1 or more and 5 or less amino acids, and further preferably 1 or moreand 3 or less amino acids) are mutated.

The above amino acid mutation includes deletion, substitution, insertionor addition of amino acids.

A method of introducing the mutation into an amino acid sequenceincludes a method of, for example, introducing mutations into anucleotide sequence encoding the amino acid sequence. The method ofintroducing mutations into a nucleotide sequence is described later.

The protein (c) contains an amino acid sequence of the protein (a) or(b) as part of the amino acid sequence thereof. Among the amino acidsequence composing the protein (c), specific examples of sequences otherthan the amino acid sequences of the protein (a) or (b) include anarbitrary portion of the amino acid sequence set forth in SEQ ID NO: 1other than the amino acid sequence of the 115^(th) to 274^(th) aminoacids set forth in SEQ ID NO: 1, an arbitrary portion of the amino acidsequence set forth in SEQ ID NO: 14 other than the amino acid sequenceof the 128^(th) to 287^(th) amino acids set forth in SEQ ID NO: 14, anarbitrary portion of the amino acid sequence set forth in SEQ ID NO: 16other than the amino acid sequence of the 126^(th) to 285^(th) aminoacids set forth in SEQ ID NO: 16, and an amino acid sequence in whichone or several amino acids (preferably 1 or more and 10 or less aminoacids, more preferably 1 or more and 5 or less amino acids, and furtherpreferably 1 or more and 3 or less amino acids) are mutated in thesesequences. The above amino acid mutation includes deletion,substitution, insertion or addition of amino acid.

The protein (c) is preferably a protein containing the amino acidsequence of the protein (a), a protein containing the amino acidsequence of the protein (a1), a protein containing the amino acidsequence of the protein (a2), or a protein containing an amino acidsequence of any one of the proteins (a), (a1) and (a2), in which one orseveral amino acids (preferably 1 or more and 10 or less amino acids,more preferably 1 or more and 5 or less amino acids, and furtherpreferably 1 or more and 3 or less amino acids) are mutated.

Further, as the protein (c), a protein consisting of an amino acidsequence of the 36^(th) to 274^(th) amino acids set forth in SEQ ID NO:1, a protein consisting of an amino acid sequence of the 45^(th) to274^(th) amino acids set forth in SEQ ID NO: 1, a protein consisting ofan amino acid sequence of the 55^(th) to 274^(th) amino acids set forthin SEQ ID NO: 1, a protein consisting of an amino acid sequence of the65^(th) to 274^(th) amino acids set forth in SEQ ID NO: 1, a proteinconsisting of an amino acid sequence of the 75^(th) to 274^(th) aminoacids set forth in SEQ ID NO: 1, a protein consisting of an amino acidsequence of the 85^(th) to 274^(th) amino acids set forth in SEQ ID NO:1, a protein consisting of an amino acid sequence of the 95^(th) to274^(th) amino acids set forth in SEQ ID NO: 1, and a protein consistingof an amino acid sequence of the 105^(th) to 274^(th) amino acids setforth in SEQ ID NO: 1 are more preferable. Further, a protein consistingof any one of these amino acid sequences, in which one or several aminoacids (preferably 1 or more and 20 or less amino acids, more preferably1 or more and 15 or less amino acids, further preferably 1 or more and10 or less amino acids, furthermore preferably 1 or more and 5 or lessamino acids, and particularly preferably 1 or more and 3 or less aminoacids) are mutated, is also preferable. These proteins are confirmed tohave the acyl-ACP thioesterase activity by Examples described later.

Further, as the protein (c), a protein consisting of an amino acidsequence set forth in SEQ ID NO: 14, a protein consisting of an aminoacid sequence of the 49^(th) to 287^(th) amino acids set forth in SEQ IDNO: 14, a protein consisting of an amino acid sequence of the 58^(th) to287^(th) amino acids set forth in SEQ ID NO: 14, a protein consisting ofan amino acid sequence of the 78^(th) to 287^(th) amino acids set forthin SEQ ID NO: 14, a protein consisting of an amino acid sequence of the88^(th) to 287^(th) amino acids set forth in SEQ ID NO: 14, a proteinconsisting of an amino acid sequence of the 98^(th) to 287^(th) aminoacids set forth in SEQ ID NO: 14, a protein consisting of an amino acidsequence of the 108^(th) to 287^(th) amino acids set forth in SEQ ID NO:14, a protein consisting of an amino acid sequence of the 118^(th) to287^(th) amino acids set forth in SEQ ID NO: 14, and a proteinconsisting of any one of these amino acid sequences, in which one orseveral amino acids (preferably 1 or more and 20 or less amino acids,more preferably 1 or more and 15 or less amino acids, further preferably1 or more and 10 or less amino acids, furthermore preferably 1 or moreand 5 or less amino acids, and particularly preferably 1 or more and 3or less amino acids) are mutated, are more preferable.

Further, as the protein (c), a protein consisting of an amino acidsequence set forth in SEQ ID NO: 16, a protein consisting of an aminoacid sequence of the 35^(th) to 285^(th) amino acids set forth in SEQ IDNO: 16, a protein consisting of an amino acid sequence of the 55^(th) to285^(th) amino acids set forth in SEQ ID NO: 16, a protein consisting ofan amino acid sequence of the 85^(th) to 285^(th) amino acids set forthin SEQ ID NO: 16, and a protein consisting of any one of these aminoacid sequences, in which one or several amino acids (preferably 1 ormore and 20 or less amino acids, more preferably 1 or more and 15 orless amino acids, further preferably 1 or more and 10 or less aminoacids, furthermore preferably 1 or more and 5 or less amino acids, andparticularly preferably 1 or more and 3 or less amino acids) aremutated, are more preferable.

The above amino acid mutation includes deletion, substitution, insertionor addition of amino acid.

Moreover, the protein (c) also preferably includes a protein consistingof an amino acid sequence formed such that a signal peptide engaging intransport or secretion of the protein is added to the amino acidsequence of the protein (a) or (b). Specific examples of addition of thesignal peptide include addition to an N terminus of chloroplast transitpeptide.

The acyl-ACP thioesterase activity of the protein of the presentinvention can be measured by, for example, introducing a fusion geneproduced by linking the acyl-ACP thioesterase gene to the downstream ofa promoter which functions in a host cell such as Escherichia coli, intoa host cell which lacks a fatty acid degradation system, culturing thethus-obtained cell under the conditions suitable for the expression ofthe introduced acyl-ACP thioesterase gene, and analyzing any changecaused thereby in the fatty acid composition of the cell or the culturedliquid by using a gas chromatographic analysis or the like.

Alternatively, the acyl-ACP thioesterase activity can be measured byintroducing a fusion gene produced by linking the acyl-ACP thioesterasegene to the downstream of a promoter which functions in a host cell suchas Escherichia coli, into a host cell, culturing the thus-obtained cellunder the conditions suitable for the expression of the introducedacyl-ACP thioesterase gene, and subjecting a disruption liquid of thecell to a reaction which uses acyl-ACPs, as substrates, preparedaccording to the method of Yuan (Yuan L, Voelker T A, Hawkins D J.“Modification of the substrate specificity of an acyl-acyl carrierprotein thioesterase by protein engineering”, Proc. Natl. Acad. Sci.U.S.A., 1995 Nov. 7; 92 (23), p. 10639-10643).

There are no particular limitations on the method for obtaining theprotein of the present invention, and the protein may be obtained bychemical techniques or genetic engineering techniques that areordinarily carried out. For example, a natural product-derived proteincan be obtained through isolation, purification and the like fromNannochloropsis gaditana. Furthermore, the protein can also beartificially synthesized based on the information for the amino acidsequence set forth in SEQ ID NO: 1, and protein synthesis may be carriedout by chemical synthesis, or a recombinant protein may also be producedby gene recombination technologies. In the case of producing arecombinant protein, the acyl-ACP thioesterase gene of the presentinvention described below can be used.

2. Acyl-ACP Thioesterase Gene

The gene of the present invention is a gene encoding any one of theproteins (a) to (c).

Specific examples of the gene encoding any one of the proteins (a) to(c) include a gene consisting of any one of DNAs (d) to (f) as follows:

-   (d) A DNA consisting of a nucleotide sequence of the 343^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   (e) A DNA consisting of a nucleotide sequence having 50% or more    identity with the nucleotide sequence of the DNA (d), and encoding a    protein having acyl-ACP thioesterase activity; and-   (f) A DNA containing the nucleotide sequence of the DNA (d) or (e),    and encoding a protein having acyl-ACP thioesterase activity.

In the DNA (e), the sequence identity of nucleotide sequence ispreferably 60% or more, more preferably 70% or more, further preferably80% or more, further more preferably 90% or more, and particularlypreferably 95% or more, in view of acyl-ACP thioesterase activity. Thesequence identity of nucleotide sequence can be calculated through themethod described above.

Specific examples of the DNA (e) include the following DNA (d1) or (d2).

-   (d1) A DNA consisting of a nucleotide sequence of the 382^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15.-   (d2) A DNA consisting of a nucleotide sequence of the 376^(th) to    858^(th) nucleotides set forth in SEQ ID NO: 17.

The nucleotide sequence set forth in SEQ ID NO: 15 is a gene encoding anovel acyl-ACP thioesterase derived from Nannochloropsis oculata. Thenucleotide sequence of the 382^(nd) to 864^(th) nucleotides set forth inSEQ ID NO: 15 has about 76% identity with the nucleotide sequence of the343^(rd) to 825^(th) nucleotides set forth in SEQ ID NO: 2.

The nucleotide sequence set forth in SEQ ID NO: 17 is a gene encoding anovel acyl-ACP thioesterase derived from Nannochloropsis granulata. Thenucleotide sequence of the 376^(th) to 858^(th) nucleotides set forth inSEQ ID NO: 17 has about 75% identity with the nucleotide sequence of the343^(rd) to 825^(th) nucleotides set forth in SEQ ID NO: 2.

The DNA (e) is preferably the DNA (d1) or (d2).

Further, the DNA (e) is also preferably a DNA consisting of a nucleotidesequence of the DNA (d) in which one or several nucleotides (preferably1 or more and 10 or less nucleotides, more preferably 1 or more and 5 orless nucleotides, and further preferably 1 or more and 3 or lessnucleotides) are mutated.

Further, the DNA (e) is also preferably a DNA consisting of a nucleotidesequence of the DNA (d1) in which one or several nucleotides (preferably1 or more and 10 or less nucleotides, more preferably 1 or more and 5 orless nucleotides, and further preferably 1 or more and 3 or lessnucleotides) are mutated.

Further, the DNA (e) is also preferably a DNA consisting of a nucleotidesequence of the DNA (d2) in which one or several nucleotides (preferably1 or more and 10 or less nucleotides, more preferably 1 or more and 5 orless nucleotides, and further preferably 1 or more and 3 or lessnucleotides) are mutated.

The above nucleotide mutation includes deletion, substitution, insertionor addition of nucleotide.

A method of introducing the mutation into a nucleotide sequence includesa method of introducing a site-specific mutation. Examples of the methodof introducing the site-specific mutation include a method of utilizingthe splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68,1989), the ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276,1995), and the Kunkel method (Kunkel, T. A., Proc. Natl. Acad. Sci. USA,1985, 82, 488). Further, commercially available kits such asSite-Directed Mutagenesis System Mutan-SuperExpress Km kit (manufacturedby Takara Bio, Inc.), Transformer TM Site-Directed Mutagenesis kit(manufactured by Clonetech Laboratories, Inc.), and KOD-Plus-Mutagenesiskit (manufactured by Toyobo Co., Ltd.) can also be utilized.Furthermore, a gene containing a desired mutation can also be obtainedby introducing a genetic mutation at random, and then performing anevaluation of the enzyme activities and a gene analysis thereof by anappropriate method.

The DNA (f) contains a nucleotide sequence of the DNA (d) or (e) as partof the nucleotide sequence thereof. Among the nucleotide sequencecomposing the DNA (f), specific examples of sequences other than thenucleotide sequence of the DNA (d) or (e) include an arbitrary portionof the nucleotide sequence set forth in SEQ ID NO: 2 other than thenucleotide sequence of the 343^(rd) to 825^(th) nucleotides set forth inSEQ ID NO: 2, an arbitrary portion of the nucleotide sequence set forthin SEQ ID NO: 15 other than the nucleotide sequence of the 382″^(d) to864^(th) nucleotides set forth in SEQ ID NO: 15, an arbitrary portion ofthe nucleotide sequence set forth in SEQ ID NO: 17 other than thenucleotide sequence of the 376^(th) to 858^(th) nucleotides set forth inSEQ ID NO: 17, and a nucleotide sequence in which one or severalnucleotides (preferably 1 or more and 10 or less nucleotides, morepreferably 1 or more and 5 or less nucleotides, and further preferably 1or more and 3 or less nucleotides) are mutated in these sequences. Theabove nucleotide mutation includes deletion, substitution, insertion oraddition of nucleotide.

The DNA (f) is preferably a DNA containing the nucleotide sequence ofthe DNA (d), a DNA containing the nucleotide sequence of the DNA (d1), aDNA containing the nucleotide sequence of the DNA (d2), or a DNAcontaining a nucleotide sequence of any one of the the DNAs (d), (d1)and (d2), in which one or several nucleotides (preferably 1 or more and10 or less nucleotides, more preferably 1 or more and 5 or lessnucleotides, and further preferably 1 or more and 3 or less nucleotides)are mutated.

Further, as the DNA (f), a DNA consisting of a nucleotide sequence ofthe 106^(th) to 825^(th) nucleotides set forth in SEQ ID NO: 2, a DNAconsisting of a nucleotide sequence of the 133^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, a DNA consisting of a nucleotidesequence of the 163^(rd) to 825^(th) nucleotides set forth in SEQ ID NO:2, a DNA consisting of a nucleotide sequence of the 193^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, a DNA consisting of a nucleotidesequence of the 223^(rd) to 825^(th) nucleotides set forth in SEQ ID NO:2, a DNA consisting of a nucleotide sequence of the 253^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, a DNA consisting of a nucleotidesequence of the 283^(rd) to 825^(th) nucleotides set forth in SEQ ID NO:2, and a DNA consisting of a nucleotide sequence of the 313^(rd) to825^(th) nucleotides set forth in SEQ ID NO: 2 are more preferable.Further, a DNA consisting of any one of these nucleotide sequences, inwhich one or several nucleotides (preferably 1 or more and 20 or lessnucleotides, more preferably 1 or more and 15 or less nucleotides,further preferably 1 or more and 10 or less nucleotides, furthermorepreferably 1 or more and 5 or less nucleotides, and particularlypreferably 1 or more and 3 or less nucleotides) are mutated, is alsopreferable. The proteins encoded by these DNAs are confirmed to have theacyl-ACP thioesterase activity by Examples described later.

Further, as the DNA (f), a DNA consisting of a nucleotide sequence setforth in SEQ ID NO: 15, a DNA consisting of a nucleotide sequence of the145^(th) to 864^(th) nucleotides set forth in SEQ ID NO: 15, a DNAconsisting of a nucleotide sequence of the 172^(nd) to 864^(th)nucleotides set forth in SEQ ID NO: 15, a DNA consisting of a nucleotidesequence of the 232^(nd) to 864^(th) nucleotides set forth in SEQ ID NO:15, a DNA consisting of a nucleotide sequence of the 262^(nd) to864^(th) nucleotides set forth in SEQ ID NO: 15, a DNA consisting of anucleotide sequence of the 292^(nd) to 864^(th) nucleotides set forth inSEQ ID NO: 15, a DNA consisting of a nucleotide sequence of the 322^(nd)to 864^(th) nucleotides set forth in SEQ ID NO: 15, a DNA consisting ofa nucleotide sequence of the 352^(nd) to 864^(th) nucleotides set forthin SEQ ID NO: 15, and a DNA consisting of any one of these nucleotidesequences, in which one or several nucleotides (preferably 1 or more and20 or less nucleotides, more preferably 1 or more and 15 or lessnucleotides, further preferably 1 or more and 10 or less nucleotides,furthermore preferably 1 or more and 5 or less nucleotides, andparticularly preferably 1 or more and 3 or less nucleotides) aremutated, are more preferable.

Further, as the DNA (f), a DNA consisting of a nucleotide sequence setforth in SEQ ID NO: 17, a DNA consisting of a nucleotide sequence of the103^(rd) to 858^(th) nucleotides set forth in SEQ ID NO: 17, a DNAconsisting of a nucleotide sequence of the 163^(rd) to 858^(th)nucleotides set forth in SEQ ID NO: 17, a DNA consisting of a nucleotidesequence of the 253^(rd) to 858^(th) nucleotides set forth in SEQ ID NO:17, and a DNA consisting of any one of these nucleotide sequences, inwhich one or several nucleotides (preferably 1 or more and 20 or lessnucleotides, more preferably 1 or more and 15 or less nucleotides,further preferably 1 or more and 10 or less nucleotides, furthermorepreferably 1 or more and 5 or less nucleotides, and particularlypreferably 1 or more and 3 or less nucleotides) are mutated, are morepreferable.

The above nucleotide mutation includes deletion, substitution, insertionor addition of nucleotide.

Moreover, the DNA (f) also preferably includes a DNA consisting of anucleotide sequence formed such that a nucleotide sequence encoding asignal peptide engaging in transport or secretion of the protein isadded to the nucleotide sequence of the protein (d) or (e). Specificexamples of the signal peptide to be added thereto include the proteinsdescribed in the protein (c).

A method of obtaining the acyl-ACP thioesterase gene of the presentinvention is not particularly limited, and the thioesterase gene can beobtained by ordinary genetic engineering techniques. For example, thethioesterase gene of the present invention can be obtained by artificialsynthesis based on the amino acid sequence set forth in SEQ ID NO: 1 orthe nucleotide sequence set forth in SEQ ID NO: 2. The artificialsynthesis of a gene can be achieved by utilizing the services such asInvitrogen, Inc. Furthermore, the gene can also be obtained by cloningfrom Nannochloropsis gaditana. The cloning can be carried out by, forexample, the methods described in Molecular Cloning—A LABORATORY MANUALTHIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring HarborLaboratory Press (2001)] and the like.

3. Transformant (1) First Embodiment

The transformant of the first embodiment of the present invention isobtained by introducing the acyl-ACP thioesterase gene of the presentinvention or a recombinant vector containing the gene into a host.

The introduction of the acyl-ACP thioesterase gene into a host can becarried out according to an ordinary genetic engineering method.Specifically, the transformant can be produced by preparing a vectorcapable of expressing the acyl-ACP thioesterase gene of the presentinvention in a host cell, introducing this vector into a host cell totransform the host cell.

The host cell used for the transformant is not particularly limited, andexamples of the host cell include microorganisms, plants or animals. Inthe present invention, microorganisms include microalgae. Among these,microorganisms and plants are preferable, and microorganisms are morepreferable, from the viewpoints of production efficiency and theusability of lipids.

As the microorganisms for the host cell, prokaryotes and eukaryotes canbe used. Prokaryotes include microorganisms which belong to the genusEscherichia or microorganisms which belong to the genus Bacillus.Eukaryotes include yeast or filamentous fungi. Among them, from theviewpoint of the productivity of lipids, Escherichia coli, Bacillussubtilis, Rhodosporidium toruloides, and Mortierella sp. are preferable,and Escherichia coli is more preferable.

As the microorganisms for the host cell, microalgae are also preferable.As microalgae, from a viewpoint of establishment of a gene recombinationtechnique, algae belonging to the genus Chlamydomonas, algae belongingto the genus Chlorella, algae belonging to the genus Phaeodactylum, andalgae belonging to the genus Nannochloropsis are preferable, and algaebelonging to the genus Nannochloropsis are more preferable. Specificexamples of the algae belonging to the genus Nannochloropsis includeNannochloropsis oculata, Nannochloropsis paditana, Nannochloropsissalina, Nannochloropsis oceanica, Nannochloropsis atomus,Nannochloropsis maculata, Nannochloropsis granulata, and Nannochloropsissp. Among them, from the viewpoint of the productivity of lipids,Nannochloropsis oculata or Nannochloropsis qaditana is preferable, andNannochloropsis oculata is more preferable.

As the plants for the host cell, from the viewpoint of a lipid contentof seeds, Arabidopsis thaliana, rapeseed, Cocos nucifera, palm, cuphea,and Jatropha curcas are preferable, and Arabidopsis thaliana is morepreferable.

A vector for use as the expression vector may be any vector capable ofintroducing the acyl-ACP thioesterase gene of the present invention intoa host cell, and expressing the gene in the host cell. For example, avector which has expression regulation regions such as a promoter and aterminator in accordance with the type of the host cell to be used, andhas a replication initiation point, a selection marker or the like, canbe used. Furthermore, the vector may also be a vector capable ofself-proliferation and self-replication outside the chromosome, such asa plasmid, or may also be a vector which is incorporated into thechromosome.

Specific examples of the vector include, in the case of using amicroorganism as the host cell, pBluescript II SK(−) (manufactured byStratagene Corp.), pUC-based vector (manufactured by Takara Shuzo Co.,Ltd.), a pET-based vector (manufactured by Takara Bio, Inc.), apGEX-based vector (manufactured by GE Healthcare, Inc.), a pCoid-basedvector (manufactured by Takara Bio, Inc.), pHY300PLK (manufactured byTakara Bio, Inc.), pUB110 (Mckenzie, T. et al., (1986), Plasmid 15(2);p. 93-103), pBR322 (manufactured by Takara Bio, Inc.), pRS403(manufactured by Stratagene Corp.), and pMW218/219 (manufactured byNippon Gene Co., Ltd.). Particularly, in the case of using Escherichiacoli as the host cell, pBluescript II SK(−) or pMW218/219 is preferablyused.

When the algae is used as the host cell, specific examples of the vectorinclude P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center),pPha-T1 (see Non-Patent Literature 2 described above) and pJET1(manufactured by COSMO BIO co., ltd.). In particular, in the case ofusing the algae belonging to the genus Nannochloropsis as the host cell,pPha-T1 or pJET1 is preferably used. Moreover, when the host cell is thealgae belonging to the genus Nannochloropsis, the host cell can betransformed, with referring to the method described in the literature,Oliver Kilian, et al., Proceedings of the National Academy of Sciencesof the United States of America, Dec. 27; 108(52), 2011, by using theDNA fragment consisting of the gene of the present invention, a promoterand a terminator. Specific examples of this DNA fragment include aPCR-amplified DNA fragment and a restriction enzyme-cut DNA fragment.

In the case of using a plant cell as the host cell, examples of thevector include a pRI-based vector (manufactured by Takara Bio, Inc.), apBI-based vector (manufactured by Clontech Laboratories, Inc.), and anIN3-based vector (manufactured by Inplanta Innovations, Inc.).Particularly, in the case of using Arabidopsis thaliana as the hostcell, a pRI-based vector or a pBI-based vector is preferably used.

The expression regulation regions such as a promoter and a terminator,and the selection marker are not particularly limited, and can beappropriately selected from ordinarily used promoters, markers and thelike in accordance with the type of the host cell to be used.

Specific examples of the promoter include lac promoter, trp promoter,tac promoter, trc promoter, T7 promoter, SpoVG promoter, cauliflowermosaic virus 35S RNA promoter, promoters for housekeeping genes such astubulin, actin and ubiquitin, rapeseed-derived Napin gene promoter,plant-derived Rubisco promoter, and a promoter of aviolaxanthin/(chlorophyll a)-binding protein gene derived from the genusNannochloropsis.

Examples of the selection marker include drug resistance genes such asantibiotic resistance genes (e.g. an ampicillin resistance gene, achloramphenicol resistance gene, an erythromycin resistance gene, aneomycin resistance gene, a kanamycin resistance gene, a spectinomycinresistance gene, a tetracycline resistance gene, a blasticidin Sresistance gene, a bialaphos resistance gene, a zeocin resistance gene,a paromomycin resistance gene, and a hygromycin resistance gene).Further, it is also possible to use a deletion of an auxotrophy-relatedgene or the like as the selection marker.

A vector for transformation can be constructed by introducing theacyl-ACP thioesterase gene of the present invention into theabove-described vector according to an ordinary technique such asrestriction enzyme treatment or ligation.

The method for transformation is not particularly limited as long as itis a method capable of introducing a target gene into a host cell. Forexample, a method of using calcium ion, a general competent celltransformation method (J. Bacterial. 93, 1925 (1967)), a protoplasttransformation method (Mol. Gen. Genet. 168, 111 (1979)), anelectroporation method (FEMS Microbiol. Lett. 55, 135 (1990)), an LPtransformation method (T. Akamatsu and J. Sekiguchi, Archives ofMicrobiology, 1987, 146, p. 353-357; T. Akamatsu and H. Taguchi,Bioscience, Biotechnology, and Biochemistry, 2001, 65, 4, p. 823-829)and the like, can be used. When the host is the algae belonging to thegenus Nannochloropsis, transformation can also be performed by applyingthe electroporation method described in Randor Radakovits, et al.,Nature Communications, DO1:10.1038/ncomms1688, 2012.

The selection of a transformant having a target gene fragment introducedtherein can be carried out by using the selection marker or the like.For example, the selection can be carried out by using an indicatorwhether a transformant acquires the drug resistance as a result ofintroducing a vector-derived drug resistance gene into a host celltogether with a target DNA fragment. Further, the introduction of atarget DNA fragment can also be confirmed by PCR using a genome as atemplate.

The transformant of the present embodiment can efficiently produce afatty acid having a specific number of carbon atoms (chain length) andunsaturated bonds, and can produce improved amount of lipids. Theability to produce fatty acids of the transformant can be measured bythe method used in the Examples.

(2) Second Embodiment

The transformant of the second embodiment of the present invention is atransformant in which, in a host cell having the acyl-ACP thioesterasegene of the present invention, the gene is subjected to deletion,mutation or repression of gene expression. The transformant can beobtained by deleting, mutating or repressing the acyl-ACP thioesterasegene of the present invention in the host cell.

The host cell of the transformant of this embodiment only needs to havethe acyl-ACP thioesterase gene of the present invention. For example,microorganisms, plants or animals can be used as the host cell. Amongthese, microorganisms are preferable, and microalgae are morepreferable, from the viewpoint of the productivity of lipids.

As the microalgae, from the viewpoint of the productivity of lipids,algae belonging to the genus Nannochloropsis are preferable. Specificexamples of the algae belonging to the genus Nannochloropsis includeNannochloropsis oculata, Nannochloropsis gaditana, Nannochloropsissalina, Nannochloropsis oceanica, Nannochloropsis atomus,Nannochloropsis maculata, Nannochloropsis granulata, and Nannochloropsissp. Among them, from the viewpoint of the productivity of lipids,Nannochloropsis oculata or Nannochloropsis gaditana is preferable, andNannochloropsis oculata is more preferable.

The deletion, mutation or repression of the acyl-ACP thioesterase geneof the present invention from a host genome can be conducted by a methodof partially or wholly removing a target gene from a genome, replacingthe target gene by other genes, inserting other DNA fragments into thetarget gene, or providing mutation in an active site, asubstrate-binding site, or a transcription or translation initiationregion of the target gene.

The above method of deletion, mutation or repression of gene expressioncan employ homologous recombination techniques. For example, a linearDNA fragment containing an upstream and downstream regions of a targetgene in a host genome but containing no target gene is constructed by amethod such as PCR, and the resultant DNA fragment is incorporated intoa host cell to cause double crossover homologous recombination on theside upstream and downstream of the target gene of the host genome, andthen the target gene on the genome can be deleted or substituted forother gene fragment. Moreover, a target gene into which mutation such asnucleotide substitution and nucleotide insertion is introduced isconstructed by a method such as PCR, and the resulting gene isincorporated into a host cell to cause double crossover homologousrecombination in two regions outside the mutation site in the targetgene of the host genome, and then a function of the target gene on thegenome can be deteriorated or lost. Moreover, a cyclic recombinantplasmid is prepared by introducing a DNA fragment partially containing atarget gene into a suitable plasmid vector, and the resultant plasmid isincorporated into a host cell to cause homologous recombination in partof region of the target gene on the host genome and to split the targetgene of the host genome, and then a function of the target gene can bedeteriorated or lost.

The method of deletion, mutation or repression of gene expression of atarget gene using homologous recombination can be conducted withreference to literature such as Besher et al., Methods in molecularbiology 47, pp. 291-302, 1995. In particular, when the host cell is thealgae belonging to the genus Nannochloropsis, with referring to theliterature such as Oliver Kilian, et al., Proceedings of the NationalAcademy of Sciences of the United States of America, Dec. 27; 108(52),2011, a specific gene in the genome can be deleted or disrupted by ahomologous recombination method.

The selection of transformants with deletion or the like of the targetgene can be made by a method of extracting genome DNA from thetransformant and performing PCR to amplify a region containing thetarget gene, a southern blotting method using a DNA probe to be bondedwith the target gene region, or the like.

With regard to the transformant of this embodiment, the acyl-ACPthioesterase gene of the present invention does not function. Therefore,the fatty acid composition of the lipid produced is considered to changefrom the composition original to the host. More specifically, thetransformant can produce a lipid in which the fatty acid composition ismodified.

4. Method of Producing Lipid

The transformant of the present invention is used for the productionmethod of the present invention. The production method of the presentinvention contains culturing the transformant in a medium, andcollecting a lipid from the resulting cultured product. In the presentinvention, the culturing of a transformant includes culturing of amicroorganism, algae, animal or plant, or a cell or tissue thereof, andalso cultivating of a plant in soil or the like. Moreover, the culturedproduct includes a medium used for culture, and a transformant subjectedto cultivation or the like.

The medium and culture condition can be selected in accordance with thetype of the host cell for transformation, and any appropriate preferredmedium and condition can be employed. Further, from the viewpoint of theproductivity of lipids, substrates for thioesterase or precursorsubstances participating in the fatty acid biosynthesis, such asglycerol, acetic acid or malonic acid, may be added to the medium.

For instance, in the case of using Escherichia coli as the host cell fortransformation, culture may be carried out in LB medium or OvernightExpress Instant TB Medium (manufactured by Novagen, Inc.) at 30° C. to37° C. for half a day to 1 day. In the case of using Arabidopsisthaliana as the host cell for transformation, growth may be carried outunder the temperature conditions of 20° C. to 25° C., by continuouslyirradiating white light or under white light illumination conditions ofa light period of 16 hours and a dark period of 8 hours, for one to twomonths.

When the host cell of the transformation is the algae, the followingculture media and culture conditions can be applied.

As the culture medium, a medium based on natural seawater or artificialseawater may be used. Alternatively, a commercially available culturemedium may also be used. Specific examples of the culture medium includean f/2 medium, an ESM medium, a Daigo IMK medium, an L1 medium and anMNK medium. Above all, from viewpoints of an improvement in theproductivity of lipids and a nutritional ingredient concentration, anf/2 medium, an ESM medium or a Daigo IMK medium is preferred, an f/2medium or a Daigo IMK medium is more preferred, and an f/2 medium isfurther preferred. For growth promotion of the algae and an improvementin productivity of a medium chain fatty acid, a nitrogen source, aphosphorus source, a metal salt, vitamins, a trace metal or the like canbe appropriately added to the culture medium.

An amount of the algae to be seeded to the culture medium is notparticularly limited. In view of viability, the amount is preferably 1%to 50% (vol/vol), and more preferably 1% to 10% (vol/vol), per culturemedium. Culture temperature is not particularly limited within the rangein which the temperature does not adversely affect growth of the algae,but is ordinarily in the range of 5° C. to 40° C. From viewpoints of thegrowth promotion of the algae, the improvement in productivity of amedium chain fatty acid, and reduction of production cost, thetemperature is preferably 10° C. to 35° C., and more preferably 15° C.to 30° C.

Moreover, the algae are preferably cultured under irradiation with lightso that photosynthesis can be made. The light irradiation only needs tobe made under conditions in which the photosynthesis can be made, andartificial light or sunlight may be applied. From viewpoints of thegrowth promotion of the algae and the improvement in the productivity ofa medium chain fatty acid, irradiance during the light irradiation ispreferably in the range of 100 lx to 50,000 lx, more preferably in therange of 300 to 10,000 lx, and further preferably 1,000 lx to 6,000 lx.Moreover, an interval of the light irradiation is not particularlylimited. From the viewpoints in a manner similar to the viewpointsdescribed above, the irradiation is preferably performed under a lightand dark cycle. In 24 hours, a light period is preferably 8 of 24 hours,further preferably 10 to 18 hours, and still further preferably 12hours.

Moreover, the algae are preferably cultured in the presence of a carbondioxide-containing gas or in a culture medium containing carbonate suchas sodium hydrogen carbonate so that the photosynthesis can be made. Aconcentration of carbon dioxide in the gas is not particularly limited.From the viewpoints of the growth promotion or the improvement in theproductivity of a medium chain fatty acid, the concentration ispreferably 0.03% (which is the same degree as the concentration underatmospheric conditions) to 10%, more preferably 0.05% to 5%, furtherpreferably 0.1% to 3%, and still further preferably 0.3% to 1%. Aconcentration of the carbonate is not particularly limited. When thesodium hydrogen carbonate is used, for example, from the viewpoints ofthe growth promotion and the improvement in the productivity of a mediumchain fatty acid, the concentration is preferably 0.01 to 5% by mass,more preferably 0.05 to 2% by mass, and further preferably 0.1 to 1% bymass.

A culture time is not particularly limited, and the culture may beperformed for a long time (for example, about 150 days) so that an algabody in which the lipid is accumulated at a high concentration can growat a high concentration. From the viewpoints of the growth promotion ofthe algae, the improvement in the productivity of a medium chain fattyacid, and the reduction of production cost, a culture time is preferably3 to 90 days, more preferably 3 to 30 days, and further preferably 7 to30 days. In addition, the culture may be performed in any of aerated andagitated culture, shaking culture or static culture. From a viewpoint ofimproving air-permeability, shaking culture is preferred.

Lipids produced in the transformant is isolated or collected by anordinary method used for isolating lipid components and the like. Forexample, lipid components can be isolated and collected from thecultured product or transformant by means of filtration, centrifugation,cell disruption, gel filtration chromatography, ion exchangechromatography, chloroform/methanol extraction, hexane extraction,ethanol extraction, or the like. In the case of isolation and collectionof larger scales, lipids can be obtained by collecting oil componentsfrom the cultured product or transformant through pressing orextraction, and then performing general purification processes such asdegumming, deacidification, decoloration, dewaxing, and deodorization.After lipid components are isolated as such, the isolated lipids arehydrolyzed, and thereby fatty acids can be obtained. Specific examplesof the method of isolating fatty acids from lipid components include amethod of treating the lipid components at a high temperature of about70° C. in an alkaline solution, a method of performing a lipasetreatment, and a method of degrading the lipid components usinghigh-pressure hot water.

The production method of the present invention can be preferably used inthe production of fatty acids having 8 or more and 22 or less carbonatoms and derivatives thereof. Particularly, the production method ofthe present invention can be more preferably used in the production offatty acids having 12 or more and 20 or less carbon atoms andderivatives thereof, more preferably used in the production of fattyacids having 12 or more and 14 or less carbon atoms and derivativesthereof.

The lipids obtained by the production method and the transformant of thepresent invention can be utilized for food, as well as can be utilizedas an emulsifier incorporated into cosmetic products or the like, acleansing agent such as a soap or a detergent, a fiber treatment agent,a hair conditioning agent, a disinfectant or an antiseptic. Moreover,the lipids can also be used as raw materials of biodiesel fuels.

With regard to the embodiments described above, the present inventionalso discloses a protein, a gene, a transformant, and a method describedbelow.

<1> A protein selected from the following (a) to (c):

-   (a) A protein consisting of an amino acid sequence of the 115^(th)    to 274^(th) amino acids set forth in SEQ ID NO: 1;-   (b) A protein consisting of an amino acid sequence having 50% or    more, preferably 60% or more, more preferably 70% or more, further    preferably 80% or more, further more preferably 90% or more, and    particularly preferably 95% or more identity with the amino acid    sequence of the protein (a), and having acyl-ACP thioesterase    activity; and-   (c) A protein containing the amino acid sequence of the protein (a)    or (b), and having acyl-ACP thioesterase activity.    <2> The protein according to the above item <1>, wherein the    protein (b) is a protein (a1)) or (a2) as follows:-   (a1) A protein consisting of an amino acid sequence of the 128^(th)    to 287^(th) amino acids set forth in SEQ ID NO: 14, and-   (a2) A protein consisting of an amino acid sequence of the 126^(th)    to 285^(th) amino acids set forth in SEQ ID NO: 16.    <3> The protein according to the above item <1>, wherein the    protein (b) is a protein consisting of an amino acid sequence of the    protein (a), (a1) or (a2) in which one or several amino acids,    preferably 1 or more and 10 or less amino acids, more preferably 1    or more and 5 or less amino acids, and further preferably 1 or more    and 3 or less amino acids, are mutated, and having acyl-ACP    thioesterase activity.    <4> The protein according to the above item <1>, wherein the    protein (c) is a protein selected from the group consisting of:-   a protein consisting of an amino acid sequence of the 36^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 45^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 55^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 65^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 75^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 85^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 95^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 105^(th) to    274^(th) amino acids set forth in SEQ ID NO: 1;-   a protein consisting of an amino acid sequence of the 1^(st) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 49^(th) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 58^(th) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 78^(th) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 88^(th) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 98^(th) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 108^(th) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 118^(th) to    287^(th) amino acids set forth in SEQ ID NO: 14;-   a protein consisting of an amino acid sequence of the 1^(st) to    285^(th) amino acids set forth in SEQ ID NO: 16;-   a protein consisting of an amino acid sequence of the 35^(th) to    285^(th) amino acids set forth in SEQ ID NO: 16;-   a protein consisting of an amino acid sequence of the 55^(th) to    285^(th) amino acids set forth in SEQ ID NO: 16;-   a protein consisting of an amino acid sequence of the 85^(th) to    285^(th) amino acids set forth in SEQ ID NO: 16; and-   a protein consisting of any one of these amino acid sequences, in    which one or several amino acids (preferably 1 or more and 20 or    less amino acids, more preferably 1 or more and 15 or less amino    acids, further preferably 1 or more and 10 or less amino acids,    furthermore preferably 1 or more and 5 or less amino acids, and    particularly preferably 1 or more and 3 or less amino acids) are    mutated, and having acyl-ACP thioesterase activity.    <5> The protein according to the above item <3> or <4>, wherein the    amino acid mutation is deletion, substitution, insertion or addition    of an amino acid.    <6> A gene encoding the protein according to any one of the above    items <1> to <5>.    <7> A gene consisting of any one of the following DNAs (d) to (f):-   (d) A DNA consisting of a nucleotide sequence of the 343^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   (e) A DNA consisting of a nucleotide sequence having 50% or more,    preferably 60% or more, more preferably 70% or more, further    preferably 80% or more, further more preferably 90% or more, and    particularly preferably 95% or more identity with the nucleotide    sequence of the DNA (d), and encoding a protein having acyl-ACP    thioesterase activity; and-   (f) A DNA containing the nucleotide sequence of the DNA (d) or (e),    and encoding a protein having acyl-ACP thioesterase activity.    <8> The gene according to the above item <7>, wherein the DNA (e) is    a DNA (d1) or (d2) as follows:-   (d1) A DNA consisting of a nucleotide sequence of the 382^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15, and-   (d2) A DNA consisting of a nucleotide sequence of the 376^(th) to    858^(th) nucleotides set forth in SEQ ID NO: 17.    <9> The gene according to the above item <7>, wherein the DNA (e) is    a DNA consisting of a nucleotide sequence of the DNA (d), (d1) or    (d2) in which one or several nucleotides, preferably 1 or more and    10 or less nucleotides, more preferably 1 or more and 5 or less    nucleotides, and further preferably 1 or more and 3 or less    nucleotides are mutated, and encoding a protein having acyl-ACP    thioesterase activity.    <10> The gene according to the above item <7>, wherein the DNA (f)    is a DNA selected from the group consisting of:

a DNA consisting of a nucleotide sequence of the 106^(th) to 825^(th)nucleotides set forth in SEQ ID NO: 2;

-   a DNA consisting of a nucleotide sequence of the 133^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   a DNA consisting of a nucleotide sequence of the 163^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   a DNA consisting of a nucleotide sequence of the 193^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   a DNA consisting of a nucleotide sequence of the 223^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   a DNA consisting of a nucleotide sequence of the 253^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   a DNA consisting of a nucleotide sequence of the 283^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   a DNA consisting of a nucleotide sequence of the 313^(rd) to    825^(th) nucleotides set forth in SEQ ID NO: 2;-   a DNA consisting of a nucleotide sequence of the 1^(st) to 864^(th)    nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 145^(th) to    864^(th) nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 172^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 232^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 262^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 292^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 322^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 352^(nd) to    864^(th) nucleotides set forth in SEQ ID NO: 15;-   a DNA consisting of a nucleotide sequence of the 1^(st) to 858^(th)    nucleotides set forth in SEQ ID NO: 17;-   a DNA consisting of a nucleotide sequence of the 103^(rd) to    858^(th) nucleotides set forth in SEQ ID NO: 17;-   a DNA consisting of a nucleotide sequence of the 163^(rd) to    858^(th) nucleotides set forth in SEQ ID NO: 17;-   a DNA consisting of a nucleotide sequence of the 253^(rd) to    858^(th) nucleotides set forth in SEQ ID NO: 17; and a DNA    consisting of any one of these nucleotide sequences, in which one or    several nucleotides (preferably 1 or more and 20 or less    nucleotides, more preferably 1 or more and 15 or less nucleotides,    further preferably 1 or more and 10 or less nucleotides, furthermore    preferably 1 or more and 5 or less nucleotides, and particularly    preferably 1 or more and 3 or less nucleotides) are mutated, and    encoding a protein having acyl-ACP thioesterase activity.    <11> The gene according to the above item <9> or <10>, wherein the    nucleotide mutation is deletion, substitution, insertion or addition    of a nucleotide.    <12> A recombinant vector containing the gene according to any one    of the above items <6> to <11>.    <13> A transformant obtained by introducing the gene according to    any one of the above items <6> to <11> or the recombinant vector    according to the above item <12> into a host.    <14> A transformant, wherein, in a host having the gene according to    any one of the above items <6> to <11>, the gene is subjected to    deletion, mutation or repression of gene expression.    <15> The transformant according to the above item <13> or <14>,    wherein the host is a microorganism.    <16> The transformant according to the above item <15>, wherein the    microorganism is a microalga.    <17> The transformant according to the above item <16>, wherein the    microalga is an alga belonging to the genus Chlamydomonas, an alga    belonging to the genus Chlorella, an alga belonging to the genus    Phaeodactylum, or an alga belonging to the genus Nannochloropsis,    and preferably an alga belonging to the genus Nannochloropsis.    <18> The transformant according to the above item <17>, wherein the    alga belonging to the genus Nannochloropsis is Nannochloropsis    oculata, Nannochloropsis gaditana, Nannochloropsis salina,    Nannochloropsis oceanica, Nannochloropsis atomus, Nannochloropsis    maculata, Nannochloropsis qranulata, or Nannochloropsis sp.,    preferably Nannochloropsis oculata or Nannochloropsis gaditana, and    more preferably Nannochloropsis oculata.    <19> The transformant according to the above item <15>, wherein the    microorganism is Escherichia coli.    <20> A method of producing a lipid, containing steps of:

culturing the transformant according to any one of the above items <13>to <19> in a medium; and

collecting a lipid from the resulting cultured product.

<21> The method of producing a lipid according to the above item <20>,wherein the lipid contains fatty acids having 8 or more and 22 or lesscarbon atoms, preferably 12 or more and 20 or less carbon atoms, andmore preferably 12 or more and 14 or less carbon atoms, and derivativesthereof.<22> A method of modifying a fatty acid composition in a lipid,containing introducing the gene according to any one of the above items<6> to <11> into a host.<23> A method of enhancing productivity of a lipid, containingintroducing the gene according to any one of the above items <6> to <11>into a host.<24> A method of modifying a fatty acid composition in a lipid,containing deleting, mutating or repressing the gene according to anyone of the above items <6> to <11> in a host.

EXAMPLES

Hereinafter, the present invention will be described more in detail withreference to Examples, but the present invention is not limited thereto.

Example 1 Preparation of Escherichia coli transformant having Nga07062gene, and Production of lipid by Escherichia coli transformant 1.Preparation of Nga07062 Gene

Information on sequences of total 9052 genes of Nannochloropsis gaditanaCCMP 526 were acquired from Nannochloropsis Genome Project(http://nannochloropsis.genomeprojectsolutions-databases.com/) providedby Colorado School of Mines and Genome Project Solutions. For anNga07062 gene (gene consisting of a nucleotide sequence set forth in SEQID NO: 2) being one of the genes, a function was identified by thefollowing method.

2. Construction of Plasmid for Nga07062 Gene Expression

The Nga07062 gene was used as a template, and an Nga07062 gene fragmentconsisting of a nucleotide sequence of the 106^(th) to 825^(th)nucleotides set forth in SEQ ID NO: 2 was prepared by PCR using a pairof primers set forth in SEQ ID NO: 3 and SEQ ID NO: 4 as shown in Table1 below. Herein, the gene having the nucleotide sequence set forth inSEQ ID NO: 2 was obtained utilizing customer synthesis service of anartificial gene. Moreover, a plasmid vector pBluescriptII SK(−)(manufactured by Stratagene, Inc.) was used as a template, and thepBluescriptII SK(−) was amplified by PCR using a pair of primers setforth in SEQ ID NOs: 5 and 6 shown in Table 1 below. Then, the resultantwas subjected to digestion by restriction enzyme DpnI (manufactured byTOYOBO Co., Ltd.) treatment. These two fragments were purified using aHigh Pure PCR Product Purification Kit (manufactured by Roche AppliedScience Corporation), and then fused using an In-Fusion HD Cloning Kit(manufactured by Clontech, Inc.) to construct a plasmid Nga07062_(—)106for Nga07062 gene expression. This expression plasmid was constructed inthe form of removing an amino acid sequence of the 1^(st) to 35^(th)amino acids on a side of an N-terminus of an amino acid sequence (SEQ IDNO: 1) encoded by the Nga07062 gene, and fusing with an amino acidsequence of the 1^(st) to 29^(th) amino acids on a side of an N-terminusof a LacZ protein derived from the plasmid.

The Nga07062 gene consisting of a nucleotide sequence set forth in SEQID NO: 2 was used a template, and PCR was carried out by using pairs ofany one of primers set forth in SEQ ID NOs: 7 to 13, and a primer setforth in SEQ ID NO: 4, shown in Table 1 below, to prepare an Nga07062gene fragment consisting of a nucleotide sequence of the 163^(rd) to825^(th) nucleotides set forth in SEQ ID NO: 2, an Nga07062 genefragment consisting of a nucleotide sequence of the 193^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, an Nga07062 gene fragmentconsisting of a nucleotide sequence of the 223^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, an Nga07062 gene fragmentconsisting of a nucleotide sequence of the 253^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, an Nga07062 gene fragmentconsisting of a nucleotide sequence of the 283^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, an Nga07062 gene fragmentconsisting of a nucleotide sequence of the 313^(rd) to 825^(th)nucleotides set forth in SEQ ID NO: 2, and an Nga07062 gene fragmentconsisting of a nucleotide sequence the 343^(rd) to 825^(th) nucleotidesset forth in SEQ ID NO: 2, respectively.

Each of the resultant gene fragments was fused with the pBluescriptIISK(−) vector in a manner similar to the method described above toconstruct a plasmid Nga07062_(—)163 for Nga07062 gene expression, aplasmid Nga07062_(—)193 therefor, a plasmid Nga07062_(—)223 therefor, aplasmid Nga07062_(—)253 therefor, a plasmid Nga07062_(—)283 therefor, aplasmid Nga07062_(—)313 therefor, and a plasmid Nga07062_(—)343therefor, respectively. Herein, these expression plasmids wereconstructed in the form of removing amino acid sequences of the 1^(st)to 54^(th) amino acids, the 1^(st) to 64^(th) amino acids, the 1^(st) to74^(th) amino acids, the 1^(st) to 84^(th) amino acids, the 1^(st) to94^(th) amino acids, the 1^(st) to 104^(th) amino acids or the 1^(st) to114^(th) amino acids on the side of the N-terminus of the amino acidsequence (SEQ ID NO: 1) encoded by the Nga07062 gene, respectively, andfusing with the amino acid sequence of the 1^(st) to 29^(th) amino acidson the side of the N-terminus of the LacZ protein derived from theplasmid.

TABLE 1 Table 1 Nucleotide Sequence of Primers SEQ ID NO: 3GCGGCCGCTCTAGAGCCGGCCCGAGCACTCAGCCATC SEQ ID NO: 4ACAAAATATTAACGCCTAACTGATGTCCACCTTCTTC SEQ ID NO: 5 CTCTAGAGCGGCCGCCACCGSEQ ID NO: 6 GCGTTAATATTTTGTTAAAATTCG SEQ ID NO: 7GCGGCCGCTCTAGAGTCATCCTTCTCGATCTTGTTG SEQ ID NO: 8GCGGCCGCTCTAGAGGTAGCAGGATCATTCGTCGG SEQ ID NO: 9GCGGCCGCTCTAGAGATCGCTGGGCATACAGCAGG SEQ ID NO: 10GCGGCCGCTCTAGAGGATGAAGTAAAGTCTCCGCAG SEQ ID NO: 11GCGGCCGCTCTAGAGAATGTAGGAGGCGGCGCCCCAG SEQ ID NO: 12GCGGCCGCTCTAGAGCCCTACACGGTCACTTTTGC SEQ ID NO: 13GCGGCCGCTCTAGAGCATGATCGAGTGGACACAAAAC3. Introduction of Nga07062 Gene Expression Plasmid into Escherichiacoli

An Escherichia coli mutant strain, strain K27 (fadD88) (Overath et al,Eur. J. Biochem. 7, 559-574, 1969), was transformed by a competent celltransformation method, using each of the Nga07062 gene expressionplasmids constructed in the above. Each transformant was culturedovernight at 37° C., and each colony thus obtained was inoculated in 1mL of LBAmp liquid medium (Bacto Trypton 1%, yeast extract 0.5%, NaCl1%, and Ampicillin sodium 50 μg/mL), and then cultured overnight at 37°C. Twenty microliters of the culture fluid was inoculated to 2 mL ofOvernight Express Instant TB medium (Novagen, Inc.) and was subjected toshaking culture at 30° C. After 16 hours cultivation, lipid componentscontained in the culture fluid were analyzed by the method describedbelow in the following item 5. As a negative control, Escherichia colistrain K27 transformed with the plasmid vector pBluescriptII SK(−) wasalso subjected to the same experiment.

4. Extraction of Lipid from Escherichia coli Culture Fluid and Analysisof Fatty Acid Contained Therein

To 1 mL of the culture fluid, 50 μL of 7-pentadecanone (1 mg/mL) as aninternal standard was added, and then 0.5 mL of chloroform, 1 mL ofmethanol and 10 μL of 2N hydrochloric acid were added. The mixture wassufficiently stirred and then was left for 30 minutes. Further, 0.5 mLof chloroform and 0.5 mL of a 1.5% aqueous solution of potassiumchloride were added thereto. The mixture was stirred and centrifuged for15 minutes at 3,000 rpm, and then the chloroform layer (lower layer) wascollected with pasteur pipette. A nitrogen gas was blown onto theresultant chloroform layer to be dried into solid, 0.7 mL of 0.5 Npotassium hydroxide/methanol solution was added thereto, and theresultant mixture was kept warm at 80° C. for 30 minutes. One milliliterof 14% solution of boron trifluoride (manufactured by Sigma-Aldrich) wasadded to the sample, and the mixture was kept warm at 80° C. for 10minutes. Thereafter, 1 mL of saturated saline and 1 mL of hexane wereadded thereto, and the mixture was sufficiently stirred and then wasleft for 30 minutes at room temperature. Then, the hexane layer (upperlayer) was collected to obtain fatty acid esters.

The obtain fatty acid esters were provided for gas chromatographicanalysis. The gas chromatography was carried out under the conditions asfollows:

capillary column: DB-1 MS 30 m×200 μm×0.25 μm (J&W Scientific, Inc.),mobile layer: high purity helium,flow rate inside the column: 1.0 mL/min,temperature rise program: 100° C. (for 1 min)→10° C./min→300° C. (for 5min),equilibration time: for 1 min,injection port: split injection (split ratio: 100:1),pressure 14.49 psi, 104 mL/min,amount of injection 1 μL,cleaning vial: methanol•chloroform, anddetector temperature: 300° C.

Moreover, fatty acid ester was identified by providing the identicalsample for a gas chromatography-mass spectrometry analysis underidentical conditions.

Amounts of fatty acid methyl esters were quantitatively determined basedon the peak areas of the above gas chromatographic analysis. The peakarea corresponding to the each fatty acids was compared with that of7-pentadecanone as the internal standard, and carried out correctionsbetween the samples, and then the amount of the each fatty acids perliter of the culture fluid was calculated. Further, the total amount ofthe each fatty acids was calculated by summing the amounts of the eachfatty acids thus obtained, and ratio (weight percent) of the each fattyacids in the total amount of fatty acids was calculated.

Table 2 shows the results of measuring a ratio of each fatty acid and atotal amount of fatty acids. Herein, in Table below, the description of“Cx:y” for the fatty acid composition represents a fatty acid having “x”as the number of carbon atoms, and “y” as the number of double bonds.

TABLE 2 Total amount Introduced of fatty acids Fatty Acid Composition(%) plasmid (mg/L) C12:1 C12:0 C14:1 C14:0 C16:1 C16:0 C17:0 C18:1 C19:0pBS 149.6 0.0 0.0 0.0 4.5 2.9 49.3 26.9 9.1 7.3 Nga07062_106 155.4 0.00.0 1.2 5.2 8.2 44.6 20.6 16.3 3.4 Nga07062_163 208.7 0.0 1.2 0.8 7.91.9 48.4 28.2 5.1 6.4 Nga07062_193 284.1 0.9 1.3 4.1 7.4 14.5 41.4 14.812.9 2.8 Nga07062_223 377.1 2.4 2.6 9.0 12.1 14.9 35.0 12.8 8.7 2.7Nga07062_253 377.4 3.1 2.8 10.4 11.1 17.2 32.7 11.1 8.8 2.8 Nga07062_283373.0 2.6 2.6 9.0 12.3 15.3 34.7 12.5 8.1 3.0 Nga07062_313 303.2 1.9 2.27.3 11.2 12.8 37.3 15.6 8.2 3.4 Nga07062_343 245.2 0.7 1.7 3.0 9.4 8.643.2 21.1 8.0 4.2

As shown in Table 2, an increase in a total amount of fatty acids wasobserved in the transformant having the Nga07062 gene fragment incomparison with the transformant (pBS of Table 2) having the plasmidvector pBluescriptII SK(−). Moreover, the fatty acid composition changedin the transformant in comparison with the transformant having theplasmid vector pBluescriptII SK(−). In particular, ratios of the fattyacids of C12:1, C12:0, C14:1, C14:0 and C16:1 increased. From theseresults, the protein encoded by the Nga07062 gene is thought to be theacyl-ACP thioesterase in which a specific fatty acid is cut out fromacyl-ACP. Moreover, a protein containing at least the amino acidsequence of the 115^(th) to 274^(th) amino acids set forth in SEQ ID NO:1 was found to show the acyl-ACP thioesterase activity.

Example 2 Preparation of Escherichia coli Transformant Having Acyl-ACPThioesterase Gene Derived from Nannochloropsis oculata, and Productionof Lipid by Escherichia coli Transformant

1. Preparation of Gene Encoding Acyl-ACP Thioesterase Derived fromNannochloropsis oculata (Hereinafter, Referred to as “NoTE”), andConstruction of Plasmid for NoTE Gene Expression

Total RNA of Nannochloropsis oculata NIES 2145 was extracted, andreverse transcription was performed using SuperScript (trade name) IIIFirst-Strand Synthesis SuperMix for qRT-PCR (manufactured by InvitrogenCorporation) to obtain cDNA. This cDNA was used as a template, and agene fragment consisting of a nucleotide sequence set forth in SEQ IDNO: 15 was prepared by PCR using a pair of primers set forth in SEQ IDNO: 18 and SEQ ID NO: 27 shown in Table 3 below. The resultant genefragment was subjected to cloning to the plasmid vector pBluescriptIISK(−) in a manner similar to the method in the item 2. in Example 1 toconstruct a plasmid NoTE_(—)1 for NoTE gene expression. This expressionplasmid was constructed in the form of fusing on a side of an N-terminusof 1^(st) amino acid of an amino acid sequence (SEQ ID NO: 14) encodedby the NoTE gene with the amino acid sequence of the 1^(st) to 29^(th)amino acids on the side of the N-terminus of the LacZ protein derivedfrom the plasmid.

In a similar manner, the cDNA was used a template, and PCR was carriedout by using pairs of any one of primers set forth in SEQ ID NOs: 19 to26, and a primer set forth in SEQ ID NO: 27, shown in Table 3 below, toprepare a gene fragment consisting of a nucleotide sequence of the145^(th) to 864^(th) nucleotides set forth in SEQ ID NO: 15, a genefragment consisting of a nucleotide sequence of the 172^(nd) to 864^(th)nucleotides set forth in SEQ ID NO: 15, a gene fragment consisting of anucleotide sequence of the 232^(nd) to 864^(th) nucleotides set forth inSEQ ID NO: 15, a gene fragment consisting of a nucleotide sequence ofthe 262^(nd) to 864^(th) nucleotides set forth in SEQ ID NO: 15, a genefragment consisting of a nucleotide sequence of the 292^(nd) to 864^(th)nucleotides set forth in SEQ ID NO: 15, a gene fragment consisting of anucleotide sequence of the 322^(nd) to 864^(th) nucleotides set forth inSEQ ID NO: 15, a gene fragment consisting of a nucleotide sequence the352^(nd) to 864^(th) nucleotides set forth in SEQ ID NO: 15, and a genefragment consisting of a nucleotide sequence the 382^(nd) to 864^(th)nucleotides set forth in SEQ ID NO: 15, respectively. Each of theresultant gene fragments was fused with the pBluescriptII SK(−) vectorin a manner similar to the method described above to construct a plasmidNoTE_(—)145 for NoTE gene expression, a plasmid NoTE_(—)172 therefor, aplasmid NoTE_(—)232 therefor, a plasmid NoTE_(—)262 therefor, a plasmidNoTE_(—)292 therefor, a plasmid NoTE_(—)322 therefor, a plasmidNoTE_(—)352 therefor, and a plasmid NoTE_(—)382 therefor, respectively.Herein, these expression plasmids were constructed in the form ofremoving amino acid sequences of the 1^(st) to 48^(th) amino acids, the1^(st) to 57^(th) amino acids, the 1^(st) to 77^(th) amino acids, the1^(st) to 87^(th) amino acids, the 1^(st) to 97^(th) amino acids, the1^(st) to 107^(th) amino acids, the 1^(st) to 117^(th) amino acids orthe 1^(st) to 127^(th) amino acids on the side of the N-terminus of theamino acid sequence (SEQ ID NO: 14) encoded by the NoTE gene,respectively, and fusing with the amino acid sequence of the 1^(st) to29^(th) amino acids on the side of the N-terminus of the LacZ proteinderived from the plasmid.

TABLE 3 Table 3 Nucleotide Sequence of Primers SEQ ID NO: 18GCGGCCGCTCTAGAGATGACGCCTTTGGCCTTCAC SEQ ID NO: 19GCGGCCGCTCTAGAGTCCGGCTGTTCACATAGCAC SEQ ID NO: 20GCGGCCGCTCTAGAGCTTAGAACCAGCTTCCCAGTC SEQ ID NO: 21GCGGCCGCTCTAGAGGCTGCCATTTCCCTGCCGTCG SEQ ID NO: 22GCGGCCGCTCTAGAGTGCGAGACGGCCCACGCCGGG AC SEQ ID NO: 23GCGGCCGCTCTAGAGAGACGAGGTGAGAGGAAGGC SEQ ID NO: 24GCGGCCGCTCTAGAGGATGGTGGAAAAGGCGAGGCC SEQ ID NO: 25GCGGCCGCTCTAGAGGCTACATGCAATCCATCCTTA TTC SEQ ID NO: 26GCGGCCGCTCTAGAGCATGATCGCGTCGACACCAAG C SEQ ID NO: 27ACAAAATATTAACGCCTAGCTAATATCAATTTTCTT TGG2. Introduction of NoTE Gene Expression Plasmid into Escherichia coli,Extraction of Lipid from Escherichia coli Culture Fluid, and Analysis ofFatty Acid Contained Therein

The NoTE expression plasmid was introduced into Escherichia coli in amanner similar to the method in item 3. in Example 1 to analyze a lipidin a manner similar to the method in item 4. in Example 1.

TABLE 4 Total amount Introduced of fatty acids Fatty Acid Composition(%) plasmid (mg/L) C12:1 C12:0 C14:1 C14:0 C16:1 C16:0 C17:0 C18:1 C19:0pBS 228.0 0.0 0.0 0.0 4.7 3.5 47.3 27.9 11.4 5.2 NoTE_1 348.8 1.3 2.26.9 10.2 11.8 35.9 16.9 11.9 2.9 NoTE_145 372.4 2.1 2.6 10.0 10.4 18.132.4 11.4 11.6 1.4 NoTE_172 364.7 1.1 1.4 5.4 5.5 23.1 37.6 10.1 15.10.6 NoTE_232 429.2 4.5 3.0 15.9 12.4 24.8 23.8 8.4 6.6 0.7 NoTE_262360.6 6.5 3.9 18.8 13.6 25.9 19.2 7.0 4.8 0.4 NoTE_292 460.2 2.8 2.312.0 10.8 21.7 28.9 10.6 9.1 1.8 NoTE_322 441.5 4.5 3.0 16.0 11.9 24.523.4 9.0 6.7 1.0 NoTE_352 299.2 3.7 2.2 10.2 7.2 26.3 30.9 9.1 9.8 0.7NoTE_382 349.3 1.5 1.6 5.9 5.7 21.2 37.5 11.2 14.6 0.8

As shown in Table 4, an increase in a total amount of fatty acids wasobserved in the transformant having the NoTE gene fragment in comparisonwith the transformant (pBS of Table 4) having the plasmid vectorpBluescriptII SK(−). Moreover, the fatty acid composition changed in thetransformant in comparison with the transformant having the plasmidvector pBluescriptII SK(−). In particular, ratios of the fatty acids ofC12:1, C12:0, C14:1, C14:0 and C16:1 increased. From these results, theprotein encoded by the NoTE gene is thought to be the acyl-ACPthioesterase in which a specific fatty acid is cut out from acyl-ACP.Moreover, a protein containing at least the amino acid sequence of the128^(th) to 287^(th) amino acids set forth in SEQ ID NO: 14 was found toshow the acyl-ACP thioesterase activity.

Example 3 Preparation of Escherichia coli Transformant Having Acyl-ACPThioesterase Gene Derived from Nannochloropsis granulata, and Productionof Lipid by Escherichia coli Transformant

1. Preparation of Gene Encoding Acyl-ACP Thioesterase Derived fromNannochloropsis granulata (Hereinafter, Referred to as “NgrTE”), andConstruction of Plasmid for NgrTE Gene Expression

Total RNA of Nannochloropsis granulata NIES2588 was extracted, andreverse transcription was performed using SuperScript (trade name) IllFirst-Strand Synthesis SuperMix for qRT-PCR (manufactured by InvitrogenCorporation) to obtain cDNA. This cDNA was used as a template, and agene fragment consisting of a nucleotide sequence set forth in SEQ IDNO: 17 was prepared by PCR using a pair of primers set forth in SEQ IDNO: 28 and SEQ ID NO: 33 shown in Table 5 below. The resultant genefragment was subjected to cloning to the plasmid vector pBluescriptIISK(−) in a manner similar to the method in the item 2. in Example 1 toconstruct a plasmid NgrTE_(—)1 for NgrTE gene expression. Thisexpression plasmid was constructed in the form of fusing on a side of anN-terminus of 1^(st) amino acid of an amino acid sequence (SEQ ID NO:16) encoded by the NgrTE gene with the amino acid sequence of the 1^(st)to 29^(th) amino acids on the side of the N-terminus of the LacZ proteinderived from the plasmid.

In a similar manner, the cDNA was used a template, and PCR was carriedout by using pairs of any one of primers set forth in SEQ ID NOs: 29 to32, and a primer set forth in SEQ ID NO: 33 shown in Table 5 below, toprepare a gene fragment consisting of a nucleotide sequence of the103^(rd) to 858^(th) nucleotides set forth in SEQ ID NO: 17, a genefragment consisting of a nucleotide sequence of the 183^(rd) to 858^(th)nucleotides set forth in SEQ ID NO: 17, a gene fragment consisting of anucleotide sequence of the 253^(rd) to 858^(th) nucleotides set forth inSEQ ID NO: 17, and a gene fragment consisting of a nucleotide sequenceof the 376^(th) to 858^(th) nucleotides set forth in SEQ ID NO: 17,respectively. Each of the resultant gene fragments was fused with thepBluescriptII SK(−) vector in a manner similar to the method describedabove to construct a plasmid NgrTE_(—)103 for NgrTE gene expression, aplasmid NgrTE_(—)163 therefor, a plasmid NgrTE_(—)253 therefor, and aplasmid NgrTE_(—)376 therefor, respectively. Herein, these expressionplasmids were constructed in the form of removing amino acid sequencesof the 1^(st) to 34^(th) amino acids, the 1^(st) to 54^(th) amino acids,the 1^(st) to 84^(th) amino acids or the 1^(st) to 125^(th) amino acidson the side of the N-terminus of the amino acid sequence (SEQ ID NO: 16)encoded by the NgrTE gene, respectively, and fusing with the amino acidsequence of the 1^(st) to 29^(th) amino acids on the side of theN-terminus of the LacZ protein derived from the plasmid.

TABLE 5 Table 5 Nucleotide Sequence of Primers SEQ ID NO: 28GCGGCCGCTCTAGAGATGACGCCTTTGGCCTTCAC SEQ ID NO: 29GCGGCCGCTCTAGAGTCCTCCCAGGTCACTCGACC SEQ ID NO: 30GCGGCCGCTCTAGAGACACTTAGCAACAGCTTTCC SEQ ID NO: 31GCGGCCGCTCTAGAGCTATGTGAGACGGCCCACAC SEQ ID NO: 32GCGGCCGCTCTAGAGCATGATCGCGTCGACACGAAG SEQ ID NO: 33ACAAAATATTAACGCCTAGCTAATGTCAATTTTCTT TGG2. Introduction of NgrTE Gene Expression Plasmid into Escherichia coli,Extraction of Lipid from Escherichia coli Culture Fluid, and Analysis ofFatty Acid Contained Therein

The NgrTE expression plasmid was introduced into Escherichia coli in amanner similar to the method in item 3. in Example 1 to analyze a lipidin a manner similar to the method in item 4. in Example 1.

TABLE 6 Total amount Introduced of fatty acids Fatty Acid Composition(%) plasmid (mg/L) C12:1 C12:0 C14:1 C14:0 C16:1 C16:0 C17:0 C18:1 C19:0pBS 181.4 0.0 0.0 0.0 6.4 2.5 48.1 28.3 5.9 8.7 NgrTE_1 317.1 2.1 3.99.1 14.9 10.1 33.5 14.2 6.9 5.1 NgrTE_103 344.0 2.8 4.0 12.3 14.1 14.529.7 11.2 7.9 3.3 NgrTE_163 328.4 2.2 4.1 9.4 14.9 9.9 32.9 14.6 7.0 4.9NgrTE_253 414.1 4.5 4.1 16.5 12.6 19.3 24.3 8.9 7.3 2.6 NgrTE_376 296.41.4 2.1 6.6 9.9 9.9 36.5 17.7 8.1 7.9

As shown in Table 6, an increase in a total amount of fatty acids wasobserved in the transformant having the NgrTE gene fragment incomparison with the transformant (pBS of Table 6) having the plasmidvector pBluescriptII SK(−). Moreover, the fatty acid composition changedin the transformant in comparison with the transformant having theplasmid vector pBluescriptII SK(−). In particular, ratios of the fattyacids of C12:1, C12:0, C14:1, C14:0 and C16:1 increased.

Example 4 Preparation of Escherichia coli Transformant Having MutatedNoTE Gene, and Production of Lipid by Escherichia coli Transformant 1.Preparation of Mutated NoTE Gene, and Construction of Plasmid forMutated NoTE Gene Expression

A gene sequence (SEQ ID NO: 35) encoding a mutated NoTE set forth in SEQID NO: 34 was prepared using custom synthesis service of an artificialgene. The amino acid sequence of the 128^(th) to 287^(th) amino acidsset forth in SEQ ID NO: 34 shows about 84% identity with the amino acidsequence of the 115^(th) to 274^(th) amino acids set forth in SEQ ID NO:1.

This gene fragment was used as a template, and a gene fragmentconsisting of a nucleotide sequence set forth in SEQ ID NO: 35 wasprepared by PCR using a pair of primers set forth in SEQ ID NO: 22 shownin Table 3 above and SEQ ID NO: 36 and shown in Table 7 below. Theresultant gene fragment was subjected to cloning to a plasmid vectorpBluescriptII SK(−) in a manner similar to the method in item 2. inExample 1 to construct a plasmid NoTE_(—)262-mutant for mutated NoTEgene expression. This expression plasmid was constructed in the form ofremoving an amino acid sequence of the 1^(st) to 87^(th) amino acids ona side of an N-terminus of an amino acid sequence (SEQ ID NO: 34)encoded by a mutated NoTE gene, and further fusing with the amino acidsequence of the 1^(st) to 29^(th) amino acids on the side of theN-terminus of the LacZ protein derived from the plasmid.

TABLE 7 Table 7 Nucleotide Sequence of Primer SEQ ID NO: 36ACAAAATATTAACGCCTAGCTAGTAGCAATTTTCC2. Introduction of Mutated NoTE Gene Expression Plasmid into Escherichiacoli, Extraction of Lipid from Escherichia coli Culture Fluid, andAnalysis of Fatty Acid Contained Therein

The mutated-NoTE expression plasmid was introduced into Escherichia coliin a manner similar to the method in item 3. in Example 1 to analyze alipid in a manner similar to the method in item 4. in Example 1.

TABLE 8 Total amount Introduced of fatty acids Fatty Acid Composition(%) plasmid (mg/L) C12:1 C12:0 C14:1 C14:0 C16:1 C16:0 C17:0 C18:1 C19:0pBS 203.8 0.0 0.0 0.0 5.3 1.6 46.3 29.2 4.3 13.2 NoTE_262-mutant 511.95.5 5.5 7.7 19.7 13.5 27.9 9.4 6.3 4.5

As shown in Table 8, an increase in a total amount of fatty acids wasobserved in the transformant having the mutated-NoTE gene fragment incomparison with the transformant (pBS of Table 8) having the plasmidvector pBluescriptII SK(−). Moreover, the fatty acid composition changedin the transformant in comparison with the transformant having theplasmid vector pBluescriptII SK(−). In particular, ratios of the fattyacids of C12:1, C12:0, C14:1, C14:0 and C16:1 increased. From theseresults, the mutated-NoTE was found to show the acyl-ACP thioesteraseactivity.

Example 5 Preparation of Nannochloropsis Transformant Having NoTE Gene,and Production of Lipid by Nannochloropsis Transformant 1. Constructionof Plasmid for NoTE Gene Expression

The cDNA of Nannochloropsis oculata NIES 2145 was used as a template,and PCR using a pair of primers set forth in SEQ ID NO: 37 and SEQ IDNO: 38 shown in Table 9 below was carried out to prepare a NoTE genefragment consisting of a nucleotide sequence of the 292^(nd) to 864^(th)nucleotides set forth in SEQ ID NO: 15.

A VCP1 promoter sequence (SEQ ID NO: 54), a VCP chloroplast transitpeptide sequence (SEQ ID NO: 55) and a VCP1 terminator sequence (SEQ IDNO: 56) were artificially synthesized based on the complete cds sequence(Accession number: JF957601.1) of the VCP1 (violaxanthin/(chlorophylla)-binding protein) gene of Nannochloropsis sp. W2J3B registered inGenBank. The thus-synthesized DNA fragment was used as a template, andPCR was carried out using a pair of primers set forth in SEQ ID NO: 39and SEQ ID NO: 40, a pair of primers set forth in SEQ ID NO: 41 and SEQID NO: 42, and a pair of primers set forth in SEQ ID NO: 43, and SEQ IDNO: 44 as shown in Table 9 below to prepare the VCP1 promoter sequence,the VCP1 chloroplast transit peptide sequence and the VCP1 terminatorsequence, respectively. Moreover, a plasmid vector pUC19 (manufacturedby TAKARA BIO Inc.) was used as a template, and PCR using a pair ofprimers set forth in SEQ ID NO: 45 and SEQ ID NO: 46 shown in Table 9below was carried out to amplify the plasmid vector pUC19.

The NoTE gene fragment, the VCP1 promoter sequence, the VCP1 chloroplasttransit peptide sequence and the VCP1 terminator sequence obtained asdescribed above were fused with the plasmid vector pUC19 in a mannersimilar to the method in item 2. in Example 1 to construct an expressionplasmid NoTE_(—)292_Nanno in Nannochloropsis. This expression plasmidconsisted of the pUC19 vector sequence and an insert sequence (SEQ IDNO: 57; hereinafter, referred to as “fragment for NoTE gene expression”)in which the VCP1 promoter sequence, the VCP1 chloroplast transitpeptide sequence, the NoTE gene fragment and the VCP1 terminatorsequence were linked in this order.

2. Construction of Plasmid for Zeocin Resistance Gene Expression inNannochloropsis

A Zeocin resistance gene (SEQ ID NO: 58), and a tubulin promotersequence (SEQ ID NO: 59) derived from Nannochloropsis gaditana CCMP 526described in literature (Randor Radakovits, et al., NatureCommunications, DO1:10.1038/ncomms1688, 2012) were artificiallysynthesized. The thus-synthesized DNA fragment was used as a template,and PCR was carried out using a pair of primers set forth in SEQ ID NO:47 and SEQ ID NO: 48, and a pair of primers set forth in SEQ ID NO: 49and SEQ ID NO: 50, to prepare a Zeocin resistance gene and a tubulinpromoter sequence, respectively. These amplified fragments were fusedwith the VCP1 terminator sequence and the amplified fragment of aplasmid vector pUC19 prepared in item 1. as described above in a mannersimilar to the method in item 2. in Example 1 to construct a Zeocinresistance gene expression plasmid. This expression plasmid consisted ofa pUC19 vector sequence, and an insert sequence (SEQ ID NO: 60;hereinafter, referred to as “fragment for Zeocin resistance geneexpression”) in which the tubulin promoter sequence, the Zeocinresistance gene, and the VCP1 terminator sequence were linked in thisorder.

3. Introduction of Fragment for NoTE Gene Expression intoNannochloropsis

The expression plasmid NoTE_(—)292_Nanno was used as a template, and PCRwas carried out using a pair of primers set forth in SEQ ID NO: 51 andSEQ ID NO: 52 shown in Table 9 below to amplify a fragment for NoTE geneexpression (SEQ ID NO: 57) in the plasmid. Moreover, the plasmid forZeocin resistance gene expression was used as a template, and PCR wascarried out using a pair of primers set forth in SEQ ID NO: 52 and SEQID NO: 53 to amplify a fragment for Zeocin resistance gene expression(SEQ ID NO: 60). Both of amplified fragments were purified using a HighPure PCR Product Purification Kit (manufactured by Roche Applied ScienceCorporation). Herein, sterilized water was used for elution uponpurification without using an elution buffer included in the kit.

About 10⁹ cells of Nannochloropsis oculata NIES 2145 were washed with a384 mM sorbitol solution to completely remove a salt, and the resultantwas used as a host cell of transformation. The amplified fragment forNoTE gene expression (SEQ ID NO: 57) and fragment for Zeocin resistancegene expression (SEQ ID NO: 60) as described above were mixed by about500 ng for each with the host cell, and electroporation was carried outunder conditions of 50 μF, 500Ω and 2,200 v/2 mm. After 24 hourscultivation in an f/2 liquid medium, the resultant material wasinoculated in an f/2 agar medium containing 2 μg/mL of Zeocin, andcultured for two to three weeks under 12 h/12 h light-dark conditions at25° C. under an atmosphere of 0.3% CO₂. A transformant containing thefragment for NoTE gene expression (SEQ ID NO: 57) was selected from theresultant colonies by a PCR method. The thus-selected strain was seededto 20 mL of a culture medium (hereinafter, referred to as “N15P5medium”) in which a nitrogen concentration in the f/2 medium wasreinforced 15 times, and a phosphorus concentration therein wasreinforced 5 times, and subjected to shaking culture for four weeksunder the 12 h/12 h light-dark conditions at 25° C. under the atmosphereof 0.3% CO₂ (preculture). Then, 2 mL of the preculture fluid wassubcultured to 18 mL of the N15P5 medium, and subjected to shakingculture for two weeks under the 12 h/12 h light-dark conditions at 25°C. under the atmosphere of 0.3% CO₂. In addition, as a negative control,an experiment was also conducted on NIES 2145 being wild-type.

TABLE 9 Table 9 Nucleotide Sequence of Primers SEQ ID NO: 37CGCGGTGTTGCGCGCAGACGAGGTGAGAGGAAGGC SEQ ID NO: 38CTCTTCCACAGAAGCCTAGCTAATATCAATTTTCTT TGG SEQ ID NO: 39CGAGCTCGGTACCCGGGCGGTCTTTTGTCCTTTCCT C SEQ ID NO: 40AATCTGCTCGGAGGGGAGGATC SEQ ID NO: 41CCCTCCGAGCAGATTATGAAGACCGCCGCTCTCCTC SEQ ID NO: 42GCGCGCAACACCGCGGGTGCGGGAGAAC SEQ ID NO: 43 GCTTCTGTGGAAGAGCCAGTGSEQ ID NO: 44 ACTCTAGAGGATCCCCTGATCTTGTCCATCTCGTG SEQ ID NO: 45GGGATCCTCTAGAGTCGACC SEQ ID NO: 46 CGGGTACCGAGCTCGAATTC SEQ ID NO: 47CTTTTTTGTGAAGCAATGGCCAAGTTGACCAGTGCC G SEQ ID NO: 48CTCTTCCACAGAAGCTTAGTCCTGCTCCTCGGCCAC G SEQ ID NO: 49CGAGCTCGGTACCCGACTGCGCATGGATTGACCGA SEQ ID NO: 50TGCTTCACAAAAAAGACAGCTTCTTGAT SEQ ID NO: 51 GGCGGTCTTTTGTCCTTTCCTCSEQ ID NO: 52 CTGATCTTGTCCATCTCGTG SEQ ID NO: 53 ACTGCGCATGGATTGACCGA2. Extraction of Lipid from Nannochloropsis Culture Fluid, and Analysisof Fatty Acid Contained Therein

After the culture, lipid of the resultant Nannochloropsis culture fluidwas extracted and analyzed in a manner similar to the method in item 4.in Example 1. Table 10 shows the results. In addition, in Table 10, “n”represents an integer of 0 to 5. For example, when “C18:n” wasdescribed, the description represents a total of fatty acids havingcompositions of C18:0, C18:1, C18:2, C18:3, C18:4 and C18:5.

TABLE 10 Total amount of fatty acids Fatty Acid Composition (%) (mg/L)C12:0 C14:0 C16:1 C16:0 C18:n C20:n Wild type 1108 ± 169 0.1 ± 0.0 3.9 ±0.2 28.9 ± 1.0 35.5 ± 1.4 19.0 ± 0.3 12.7 ± 2.1 NoTE-Introduced 1352 ±59  0.3 ± 0.0 6.3 ± 0.1 30.6 ± 0.3 36.1 ± 0.3 16.3 ± 0.2 10.4 ± 0.4strain

As shown in Table 10, in the Nannochloropsis transformant(“NoTE-introduced strain” in Table 10) having the NoTE gene fragment, anincrease in a total amount of fatty acid was observed in comparison withthe Nannochloropsis NIES2145 (“wild-type” in Table 10) being the host.Moreover, in the transformant, ratios of fatty acids of C12:0 and C14:0significantly increased in comparison with the Nannochloropsis NIES2145(p<0.01 for all).

Having described our invention as related to the present embodiments, itis our intention that the invention not be limited by any of the detailsof the description, unless otherwise specified, but rather be construedbroadly within its spirit and scope as set out in the accompanyingclaims.

This application claims priority on Patent Application No. 2012-286058filed in Japan on Dec. 27, 2012, which is entirely herein incorporatedby reference.

1.-14. (canceled)
 15. A method of modifying a fatty acid composition ina lipid, comprising introducing a gene encoding a protein selected fromthe following (a) to (c) or a recombinant vector comprising the geneinto a host: (a) A protein consisting of an amino acid sequence of the115th to 274th amino acids set forth in SEQ ID NO: 1; (b) A proteinconsisting of an amino acid sequence having 50% or more identity withthe amino acid sequence of the protein (a), and having acyl-ACPthioesterase activity; and (c) A protein comprising the amino acidsequence of the protein (a) or (b), and having acyl-ACP thioesteraseactivity.
 16. The method according to claim 15, wherein the protein (b)is a protein (a1) or (a2) as follows: (a1) A protein consisting of anamino acid sequence of the 128th to 287th amino acids set forth in SEQID NO: 14; and (a2) A protein consisting of an amino acid sequence ofthe 126th to 285th amino acids set forth in SEQ ID NO:
 16. 17. Themethod according to claim 16, wherein the protein (b) is a proteinconsisting of an amino acid sequence of the protein (a), (a1) or (a2) inwhich 1 or more and 10 or less amino acids are mutated, and havingacyl-ACP thioesterase activity.
 18. The method according to claim 15,wherein the protein (c) is a protein selected from the group consistingof: a protein consisting of an amino acid sequence of the 36th to 274thamino acids set forth in SEQ ID NO: 1; a protein consisting of an aminoacid sequence of the 45th to 274th amino acids set forth in SEQ ID NO:1; a protein consisting of an amino acid sequence of the 55th to 274thamino acids set forth in SEQ ID NO: 1; a protein consisting of an aminoacid sequence of the 65th to 274th amino acids set forth in SEQ ID NO:1; a protein consisting of an amino acid sequence of the 75th to 274thamino acids set forth in SEQ ID NO: 1; a protein consisting of an aminoacid sequence of the 85th to 274th amino acids set forth in SEQ ID NO:1; a protein consisting of an amino acid sequence of the 95th to 274thamino acids set forth in SEQ ID NO: 1; a protein consisting of an aminoacid sequence of the 105th to 274th amino acids set forth in SEQ ID NO:1; a protein consisting of an amino acid sequence of the 1st to 287thamino acids set forth in SEQ ID NO: 14; a protein consisting of an aminoacid sequence of the 49th to 287th amino acids set forth in SEQ ID NO:14; a protein consisting of an amino acid sequence of the 58th to 287thamino acids set forth in SEQ ID NO: 14; a protein consisting of an aminoacid sequence of the 78th to 287th amino acids set forth in SEQ ID NO:14; a protein consisting of an amino acid sequence of the 88th to 287thamino acids set forth in SEQ ID NO: 14; a protein consisting of an aminoacid sequence of the 98th to 287th amino acids set forth in SEQ ID NO:14; a protein consisting of an amino acid sequence of the 108th to 287thamino acids set forth in SEQ ID NO: 14; a protein consisting of an aminoacid sequence of the 118th to 287th amino acids set forth in SEQ ID NO:14; a protein consisting of an amino acid sequence of the 1st to 285thamino acids set forth in SEQ ID NO: 16; a protein consisting of an aminoacid sequence of the 35th to 285th amino acids set forth in SEQ ID NO:16; a protein consisting of an amino acid sequence of the 55th to 285thamino acids set forth in SEQ ID NO: 16; a protein consisting of an aminoacid sequence of the 85th to 285th amino acids set forth in SEQ ID NO:16; and a protein having any one of these amino acid sequences, in which1 or more and 20 or less amino acids are mutated, and having acyl-ACPthioesterase activity.
 19. The method according to claim 15, wherein thehost is a microalga.
 20. A method of modifying a fatty acid compositionin a lipid, comprising introducing a gene consisting of any one of thefollowing DNAs (d) to (f) or a recombinant vector comprising the geneinto a host: (d) A DNA consisting of a nucleotide sequence of the 343rdto 825th nucleotides set forth in SEQ ID NO: 2; (e) A DNA consisting ofa nucleotide sequence having 50% or more identity with the nucleotidesequence of the DNA (d), and encoding a protein having acyl-ACPthioesterase activity; and (f) A DNA comprising the nucleotide sequenceof the DNA (d) or (e), and encoding a protein having acyl-ACPthioesterase activity.
 21. The method according to claim 20, wherein theDNA (e) is a DNA (d1) or (d2) as follows: (d1) A DNA consisting of anucleotide sequence of the 382nd to 864th nucleotides set forth in SEQID NO: 15, and (d2) A DNA consisting of a nucleotide sequence of the376th to 858th nucleotides set forth in SEQ ID NO:
 17. 22. The methodaccording to claim 21, wherein the DNA (e) is a DNA consisting of anucleotide sequence of the DNA (d), (d1) or (d2) in which 1 or more and10 or less nucleotides are mutated, and encoding a protein havingacyl-ACP thioesterase activity.
 23. The method according to claim 20,wherein the DNA (f) is a DNA selected from the group consisting of: aDNA consisting of a nucleotide sequence of the 106th to 825thnucleotides set forth in SEQ ID NO: 2; a DNA consisting of a nucleotidesequence of the 133rd to 825th nucleotides set forth in SEQ ID NO: 2; aDNA consisting of a nucleotide sequence of the 163rd to 825thnucleotides set forth in SEQ ID NO: 2; a DNA consisting of a nucleotidesequence of the 193rd to 825th nucleotides set forth in SEQ ID NO: 2; aDNA consisting of a nucleotide sequence of the 223rd to 825thnucleotides set forth in SEQ ID NO: 2; a DNA consisting of a nucleotidesequence of the 253rd to 825th nucleotides set forth in SEQ ID NO: 2; aDNA consisting of a nucleotide sequence of the 283rd to 825thnucleotides set forth in SEQ ID NO: 2; a DNA consisting of a nucleotidesequence of the 313rd to 825th nucleotides set forth in SEQ ID NO: 2; aDNA consisting of a nucleotide sequence of the 1st to 864th nucleotidesset forth in SEQ ID NO: 15; a DNA consisting of a nucleotide sequence ofthe 145th to 864th nucleotides set forth in SEQ ID NO: 15; a DNAconsisting of a nucleotide sequence of the 172nd to 864th nucleotidesset forth in SEQ ID NO: 15; a DNA consisting of a nucleotide sequence ofthe 232nd to 864th nucleotides set forth in SEQ ID NO: 15; a DNAconsisting of a nucleotide sequence of the 262nd to 864th nucleotidesset forth in SEQ ID NO: 15; a DNA consisting of a nucleotide sequence ofthe 292nd to 864th nucleotides set forth in SEQ ID NO: 15; a DNAconsisting of a nucleotide sequence of the 322nd to 864th nucleotidesset forth in SEQ ID NO: 15; a DNA consisting of a nucleotide sequence ofthe 352nd to 864th nucleotides set forth in SEQ ID NO: 15; a DNAconsisting of a nucleotide sequence of the 1st to 858th nucleotides setforth in SEQ ID NO: 17; a DNA consisting of a nucleotide sequence of the103rd to 858th nucleotides set forth in SEQ ID NO: 17; a DNA consistingof a nucleotide sequence of the 163rd to 858th nucleotides set forth inSEQ ID NO: 17; a DNA consisting of a nucleotide sequence of the 253rd to858th nucleotides set forth in SEQ ID NO: 17; and a DNA having any oneof these nucleotide sequences, in which 1 or more and 20 or lessnucleotides are mutated, and encoding a protein having acyl-ACPthioesterase activity.
 24. The method according to claim 20, wherein thehost is a microalga.
 25. A method of enhancing productivity of a lipid,comprising introducing a gene encoding a protein selected from thefollowing (a) to (c) or a recombinant vector comprising the gene into ahost: (a) A protein consisting of an amino acid sequence of the 115th to274th amino acids set forth in SEQ ID NO: 1; (b) A protein consisting ofan amino acid sequence having 50% or more identity with the amino acidsequence of the protein (a), and having acyl-ACP thioesterase activity;and (c) A protein comprising the amino acid sequence of the protein (a)or (b), and having acyl-ACP thioesterase activity.
 26. The methodaccording to claim 25, wherein the protein (b) is a protein (a1) or (a2)as follows: (a1) A protein consisting of an amino acid sequence of the128th to 287th amino acids set forth in SEQ ID NO: 14; and (a2) Aprotein consisting of an amino acid sequence of the 126th to 285th aminoacids set forth in SEQ ID NO:
 16. 27. The method according to claim 26,wherein the protein (b) is a protein consisting of an amino acidsequence of the protein (a), (a1) or (a2) in which 1 or more and 10 orless amino acids are mutated, and having acyl-ACP thioesterase activity.28. The protein according to claim 25, wherein the protein (c) is aprotein selected from the group consisting of: a protein consisting ofan amino acid sequence of the 36th to 274th amino acids set forth in SEQID NO: 1; a protein consisting of an amino acid sequence of the 45th to274th amino acids set forth in SEQ ID NO: 1; a protein consisting of anamino acid sequence of the 55th to 274th amino acids set forth in SEQ IDNO: 1; a protein consisting of an amino acid sequence of the 65th to274th amino acids set forth in SEQ ID NO: 1; a protein consisting of anamino acid sequence of the 75th to 274th amino acids set forth in SEQ IDNO: 1; a protein consisting of an amino acid sequence of the 85th to274th amino acids set forth in SEQ ID NO: 1; a protein consisting of anamino acid sequence of the 95th to 274th amino acids set forth in SEQ IDNO: 1; a protein consisting of an amino acid sequence of the 105th to274th amino acids set forth in SEQ ID NO: 1; a protein consisting of anamino acid sequence of the 1st to 287th amino acids set forth in SEQ IDNO: 14; a protein consisting of an amino acid sequence of the 49th to287th amino acids set forth in SEQ ID NO: 14; a protein consisting of anamino acid sequence of the 58th to 287th amino acids set forth in SEQ IDNO: 14; a protein consisting of an amino acid sequence of the 78th to287th amino acids set forth in SEQ ID NO: 14; a protein consisting of anamino acid sequence of the 88th to 287th amino acids set forth in SEQ IDNO: 14; a protein consisting of an amino acid sequence of the 98th to287th amino acids set forth in SEQ ID NO: 14; a protein consisting of anamino acid sequence of the 108th to 287th amino acids set forth in SEQID NO: 14; a protein consisting of an amino acid sequence of the 118thto 287th amino acids set forth in SEQ ID NO: 14; a protein consisting ofan amino acid sequence of the 1st to 285th amino acids set forth in SEQID NO: 16; a protein consisting of an amino acid sequence of the 35th to285th amino acids set forth in SEQ ID NO: 16; a protein consisting of anamino acid sequence of the 55th to 285th amino acids set forth in SEQ IDNO: 16; a protein consisting of an amino acid sequence of the 85th to285th amino acids set forth in SEQ ID NO: 16; and a protein having anyone of these amino acid sequences, in which 1 or more and 20 or lessamino acids are mutated, and having acyl-ACP thioesterase activity. 29.The method according to claim 25, wherein the host is a microalga.
 30. Amethod of enhancing productivity of a lipid, comprising introducing agene consisting of any one of the following DNAs (d) to (f) or arecombinant vector comprising the gene into a host: (d) A DNA consistingof a nucleotide sequence of the 343rd to 825th nucleotides set forth inSEQ ID NO: 2; (e) A DNA consisting of a nucleotide sequence having 50%or more identity with the nucleotide sequence of the DNA (d), andencoding a protein having acyl-ACP thioesterase activity; and (f) A DNAcomprising the nucleotide sequence of the DNA (d) or (e), and encoding aprotein having acyl-ACP thioesterase activity.
 31. The method accordingto claim 30, wherein the DNA (e) is a DNA (d1) or (d2) as follows: (d1)A DNA consisting of a nucleotide sequence of the 382nd to 864thnucleotides set forth in SEQ ID NO: 15, and (d2) A DNA consisting of anucleotide sequence of the 376th to 858th nucleotides set forth in SEQID NO:
 17. 32. The method according to claim 31, wherein the DNA (e) isa DNA consisting of a nucleotide sequence of the DNA (d), (d1) or (d2)in which 1 or more and 10 or less nucleotides are mutated, and encodinga protein having acyl-ACP thioesterase activity.
 33. The methodaccording to claim 30, wherein the DNA (f) is a DNA selected from thegroup consisting of: a DNA consisting of a nucleotide sequence of the106th to 825th nucleotides set forth in SEQ ID NO: 2; a DNA consistingof a nucleotide sequence of the 133rd to 825th nucleotides set forth inSEQ ID NO: 2; a DNA consisting of a nucleotide sequence of the 163rd to825th nucleotides set forth in SEQ ID NO: 2; a DNA consisting of anucleotide sequence of the 193rd to 825th nucleotides set forth in SEQID NO: 2; a DNA consisting of a nucleotide sequence of the 223rd to825th nucleotides set forth in SEQ ID NO: 2; a DNA consisting of anucleotide sequence of the 253rd to 825th nucleotides set forth in SEQID NO: 2; a DNA consisting of a nucleotide sequence of the 283rd to825th nucleotides set forth in SEQ ID NO: 2; a DNA consisting of anucleotide sequence of the 313rd to 825th nucleotides set forth in SEQID NO: 2; a DNA consisting of a nucleotide sequence of the 1st to 864thnucleotides set forth in SEQ ID NO: 15; a DNA consisting of a nucleotidesequence of the 145th to 864th nucleotides set forth in SEQ ID NO: 15; aDNA consisting of a nucleotide sequence of the 172nd to 864thnucleotides set forth in SEQ ID NO: 15; a DNA consisting of a nucleotidesequence of the 232nd to 864th nucleotides set forth in SEQ ID NO: 15; aDNA consisting of a nucleotide sequence of the 262nd to 864thnucleotides set forth in SEQ ID NO: 15; a DNA consisting of a nucleotidesequence of the 292nd to 864th nucleotides set forth in SEQ ID NO: 15; aDNA consisting of a nucleotide sequence of the 322nd to 864thnucleotides set forth in SEQ ID NO: 15; a DNA consisting of a nucleotidesequence of the 352nd to 864th nucleotides set forth in SEQ ID NO: 15; aDNA consisting of a nucleotide sequence of the 1st to 858th nucleotidesset forth in SEQ ID NO: 17; a DNA consisting of a nucleotide sequence ofthe 103rd to 858th nucleotides set forth in SEQ ID NO: 17; a DNAconsisting of a nucleotide sequence of the 163rd to 858th nucleotidesset forth in SEQ ID NO: 17; a DNA consisting of a nucleotide sequence ofthe 253rd to 858th nucleotides set forth in SEQ ID NO: 17; and a DNAhaving any one of these nucleotide sequences, in which 1 or more and 20or less nucleotides are mutated, and encoding a protein having acyl-ACPthioesterase activity.
 34. The method according to claim 30, wherein thehost is a microalga.